JP2006304779A - Plant producing hexosamine in high productivity - Google Patents
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本発明はヘキソサミン高生産形質転換植物細胞又は形質転換植物又はその子孫又はそれらの器官又はそれらの組織、発現用組換えベクター、植物抽出物、およびヘキソサミンの製造法に関する。 The present invention relates to a hexosamine high-producing transformed plant cell or transformed plant or its progeny or organ or tissue thereof, a recombinant vector for expression, a plant extract, and a method for producing hexosamine.
ヘキソサミンはヘキソースにアミノ基が導入された化合物であり、そのＮ−アセチル体が多くの糖タンパク質、糖脂質、多糖の構成成分となっている。 Hexosamine is a compound in which an amino group is introduced into hexose, and its N-acetyl form is a constituent of many glycoproteins, glycolipids, and polysaccharides.
ヘキソサミンとして産業上有用なものの一つとして、Ｎ−アセチルグルコサミン（以下、GlcNAcと略することもある）があげられる。Ｎ−アセチルグルコサミンはさわやかな甘味を有していることから、苦味を有するグルコサミンの代わりに、医薬品や食品材料としての需要が高まっている。Ｎ−アセチルグルコサミンはカニ、エビ、イカなどの甲殻類の細胞壁中のキチンの構成単位であり、これを材料として様々な方法が開発されている。 One of industrially useful hexosamines is N-acetylglucosamine (hereinafter sometimes abbreviated as GlcNAc). Since N-acetylglucosamine has a refreshing sweetness, demand for pharmaceuticals and food materials is increasing instead of glucosamine having a bitter taste. N-acetylglucosamine is a structural unit of chitin in the cell walls of crustaceans such as crabs, shrimps and squids, and various methods have been developed using this as a material.
Ｎ−アセチルグルコサミンの製造法として、キチンを酸加水分解する方法（非特許文献１、特許文献１）、酵素によりキチンを加水分解し、Ｎ−アセチルグルコサミンを遊離させる方法（特許文献２）、キチン原料にキチナーゼ生産菌の培養液を加えて培養し、Ｎ−アセチルグルコサミンを回収する方法（特許文献３）が開示されている。
As a method for producing N-acetylglucosamine, a method of hydrolyzing chitin (Non-patent
しかしながら、海洋生物のキチンを原料としてＮ−アセチルグルコサミンを得る製造法は、地球環境の悪化に伴う海洋汚染による安全性、海洋資源の乱獲による資源の枯渇が予想され、安全なキチン原料の安定した確保が難しくなる。 However, the production method for obtaining N-acetylglucosamine using chitin from marine organisms is expected to be safe due to marine pollution accompanying the deterioration of the global environment and depletion of resources due to over-exploitation of marine resources. It becomes difficult to secure.
また、クロレラウイルス感染したクロレラからＮ−アセチルグルコサミンを回収する方法（特許文献4）が開示されている。しかしながら、クロレラ細胞を用いた製造法はウイルス感染による溶菌が生じるため、工業的規模での生産には至っていない。 In addition, a method for recovering N-acetylglucosamine from chlorella infected with chlorella virus (Patent Document 4) is disclosed. However, the production method using chlorella cells does not reach production on an industrial scale because lysis occurs due to virus infection.
Ｎ−アセチルグルコサミンは、種々のグルコサミンの誘導体から成るヘキソサミン合成経路を経て生成する。このヘキソサミン合成経路は、グルタミン：フルクトース−６−リン酸アミドトランスフェラーゼ（Glutamine:fructose-6-phosphate amidotransferase、以下、GFATと略記する）により開始される。GFATは、生体内において、フルクトース−６−リン酸からグルコサミン−６−リン酸（以下、GlcN-6-Pと略することもある）への反応を触媒し、ヘキソサミン生合成経路の律速段階とされる重要な酵素である（非特許論文2）。
GFATは、動物または微生物において、ヒト、マウス、酵母および大腸菌等で見出され、これらの細胞から精製された酵素、あるいは該酵素の遺伝子クローニングで得られた組換え酵素が報告されている(非特許文献３、４、５および６)。
N-acetylglucosamine is produced through a hexosamine synthesis pathway composed of various glucosamine derivatives. This hexosamine synthesis pathway is initiated by glutamine: fructose-6-phosphate amidotransferase (hereinafter abbreviated as GFAT). GFAT catalyzes the reaction from fructose-6-phosphate to glucosamine-6-phosphate (hereinafter sometimes abbreviated as GlcN-6-P) in vivo, and the rate-limiting step of the hexosamine biosynthetic pathway It is an important enzyme (Non-Patent Paper 2).
GFAT is found in animals, microorganisms, humans, mice, yeast, E. coli, etc., and an enzyme purified from these cells, or a recombinant enzyme obtained by gene cloning of the enzyme has been reported (non-
その他、クロレラウイルス由来ＧＦＡＴを大腸菌で発現させた組換え酵素によりＮ−アセチルグルコサミンおよびグルコサミンを製造する方法が開示されている（特許文献４）。しかしながら、大腸菌を用いた製造法は細胞内にのみＮ−アセチルグルコサミンおよびグルコサミンが蓄積し、工業的規模での生産には至っていない。また、大腸菌を用いる方法では、エンドトキシン等、感染性物質の混入の危険性があり、精製などに多大なコストが必要とされ、工業化への大きな障壁となっている。 In addition, a method for producing N-acetylglucosamine and glucosamine by a recombinant enzyme in which chlorella virus-derived GFAT is expressed in Escherichia coli is disclosed (Patent Document 4). However, the production method using Escherichia coli accumulates N-acetylglucosamine and glucosamine only in cells, and has not yet produced on an industrial scale. In addition, the method using E. coli has a risk of contamination with infectious substances such as endotoxin, which requires a large amount of cost for purification and is a big barrier to industrialization.
一方、植物は、ヒトへの感染性物質を含まない安全性の高い生産系であり、動物および微生物を用いた場合に比較して、低コストの生産系である。さらに、植物は、光合成を利用して二酸化炭素と水から糖を合成する、エネルギー負荷の低い理想的な糖質生産系である。しかしながら、植物におけるGlcNAc量は極めて少ない。 On the other hand, a plant is a highly safe production system that does not contain human infectious substances, and is a low-cost production system compared to the case of using animals and microorganisms. Furthermore, plants are ideal carbohydrate production systems with low energy loads that synthesize sugar from carbon dioxide and water using photosynthesis. However, the amount of GlcNAc in plants is extremely small.
植物においてGFAT遺伝子を発現させた例として、ヘキソサミン合成経路を制御するためにトウモロコシ由来GFAT遺伝子を過剰発現させたトウモロコシ形質転換体は、カチオン性澱粉を高効率に生産することが示されているが、該形質転換体におけるヘキソサミン、特にＮ−アセチルグルコサミンおよびＮ−アセチルグルコサミン−６−リン酸（以下、GlcNAc-6-Pと略することもある）の高度な生産性との関連性は見出されていない（特許文献５）。 As an example of expressing a GFAT gene in a plant, a maize transformant overexpressing a maize-derived GFAT gene to control the hexosamine synthesis pathway has been shown to produce cationic starch with high efficiency. And the relationship between the high productivity of hexosamine, particularly N-acetylglucosamine and N-acetylglucosamine-6-phosphate (hereinafter sometimes abbreviated as GlcNAc-6-P) in the transformant. (Patent Document 5).
植物においては、ゲノム中に相同な配列を持つ外来遺伝子を植物に導入すると、サイレンシグと呼ばれるメカニズムにより、該遺伝子の発現量が減少することが知られている（非特許文献７）。上記特許文献では、サイレンシングによりGFAT遺伝子が植物内で十分に発現していないために、ヘキソサミン生産に至っていないと考えられる。
酵母をはじめとして、動物細胞、大腸菌を用いて生産する方法には特別な施設が必要で、設備投資に係るコストが問題となる。さらに、近年、動物由来物質ではBSE、鳥インフルエンザウイルス等、微生物由来物質ではエンドトキシン等、感染性物質の混入の危険性が強く懸念されている。
これらの問題点を解決する一つの手段として、植物による物質生産が挙げられる。物質生産の場としての植物は、ヒトへの感染性物質を含まない安全性の高い生産系であり、動物および微生物を用いた場合に比較して、低コストの生産系である。さらに、植物は、光合成を利用して二酸化炭素と水から糖を合成する、エネルギー負荷の低い理想的な糖質生産系である。これらの理由から、光合成エネルギーを利用して植物から有用な糖質を製造できることが可能になれば、安全、コスト、環境等の面から産業上意義は大きい。従って、本発明の目的は、植物細胞または植物体を材料として、安価かつ安全なヘキソサミンの製造法を提供することにある。
Special facilities are required for the production method using yeast, animal cells, and E. coli, and the cost of capital investment becomes a problem. Further, in recent years, there is a strong concern about the risk of contamination with infectious substances such as BSE and avian influenza virus for animal-derived substances and endotoxin for microorganism-derived substances.
One means for solving these problems is substance production by plants. A plant as a place for substance production is a highly safe production system that does not contain human infectious substances, and is a low-cost production system compared to the case of using animals and microorganisms. Furthermore, plants are ideal carbohydrate production systems with low energy loads that synthesize sugar from carbon dioxide and water using photosynthesis. For these reasons, if it becomes possible to produce useful carbohydrates from plants using photosynthetic energy, it is industrially significant in terms of safety, cost, environment, and the like. Accordingly, an object of the present invention is to provide an inexpensive and safe method for producing hexosamine using plant cells or plants as materials.
本発明者は鋭意検討した結果、由来が異なるＧＦＡＴ遺伝子を導入した植物細胞または植物体がヘキソサミンを大量に蓄積することを見出し、本発明に到達した。
上記製造法によれば、植物細胞内で発現したＧＦＡＴにより、ＧＦＡＴ反応産物であるグルコサミン−６−リン酸が生成される。さらに、植物細胞内の代謝反応により、グルコサミン−６−リン酸が代謝され、ＧｌｃＮＡｃ、GlcNAc-6-Pを含むヘキソサミンが高度に生成される。
As a result of intensive studies, the present inventor has found that plant cells or plants into which GFAT genes of different origin have been introduced accumulate a large amount of hexosamine, and have reached the present invention.
According to the above production method, glucosamine-6-phosphate, which is a GFAT reaction product, is produced by GFAT expressed in plant cells. Furthermore, glucosamine-6-phosphate is metabolized by a metabolic reaction in plant cells, and hexosamine containing GlcNAc and GlcNAc-6-P is highly produced.
すなわち、本発明はヘキソサミン高生産形質転換植物細胞又は形質転換植物又はその子孫又はそれらの器官又はそれらの組織、発現用組換えベクター、植物抽出物、およびヘキソサミンの製造法に関する。
１． 外因性GFAT（グルタミン：フルクトース−６−リン酸アミドトランスフェラーゼ）遺伝子が導入されていることを特徴とするヘキソサミン高生産形質転換植物細胞又は形質転換植物又はその子孫又はそれらの器官又はそれらの組織。
２． GFAT遺伝子が、クロレラウイルス由来ＧＦＡＴ遺伝子であることを特徴とする項１に記載のヘキソサミン高生産形質転換植物細胞又は形質転換植物又はその子孫又はそれらの器官又はそれらの組織。
３． GFAT遺伝子が、配列番号2記載の遺伝子であることを特徴とする項１又は２に記載
のヘキソサミン高生産形質転換植物細胞又は形質転換植物又はその子孫又はそれらの器官又はそれらの組織。
４． GFAT遺伝子が、配列番号2記載の遺伝子の一部、または欠失体、または付加体、ま
たは置換体であって、かつＧＦＡＴ活性を有するポリペプチドをコードする遺伝子であることを特徴とする項１又は２に記載のヘキソサミン高生産形質転換植物細胞又は形質転換植物又はその子孫又はそれらの器官又はそれらの組織。
５． クロレラウイルス由来GFAT遺伝子が導入されていることを特徴とするヘキソサミン高生産形質転換植物細胞又は形質転換植物又はその子孫又はそれらの器官又はそれらの組織。
６． 配列番号2記載のクロレラウイルス由来GFAT遺伝子が導入されていることを特徴と
するヘキソサミン高生産形質転換植物細胞又は形質転換植物又はその子孫又はそれらの器官又はそれらの組織。
７． 植物が、被子植物、裸子植物、シダ植物及びコケ植物からなる群から選ばれるいずれかの植物である、項１〜６のいずれかに記載のヘキソサミン高生産形質転換植物又はその子孫又はそれらの器官又はそれらの組織。
８． 器官が、根、茎、塊茎、葉、花器、塊根、種子及び茎頂からなる群から選ばれる１種又は２種以上の器官である、項１〜６のいずれかに記載のヘキソサミン高生産形質転換植物又はそれと同じ性質を有する子孫又はそれらの器官又はそれらの組織。
９． 組織が、表皮、師部、柔組織、木部及び維管束からなる群から選ばれる１種又は２種以上の組織である、項１〜６のいずれかに記載のヘキソサミン高生産形質転換植物又はその子孫又はそれらの器官又はそれらの組織。
１０． ヘキソサミン高生産形質転換植物又はそれと同じ性質を有する子孫又はそれらの器官又はそれらの組織を得るためのベクターであって、発現用組換えベクターに、クロレラウイルス由来ＧＦＡＴ遺伝子が導入されていることを特徴とする発現用組換えベクター。
１１． クロレラウイルス由来ＧＦＡＴ遺伝子が配列番号2記載のGFAT遺伝子であることを特徴とする、項１０記載の発現用組換えベクター。
１２． 項１〜９のいずれかに記載の植物又はそれと同じ性質を有する子孫又はそれらの器官又はそれらの組織から得られる植物抽出物。
１３． 植物抽出物がヘキソサミンであることを特徴とする項１２記載の植物抽出物。
１４． （１）外因性GFAT（グルタミン：フルクトース−６−リン酸 アミドトランスフ
ェラーゼ）遺伝子が導入されていることを特徴とする発現用組換えベクターを用いて植物細胞を形質転換する工程、
（２）形質転換して得られた形質転換体（形質転換植物細胞又は形質転換植物）を生育する工程、
（３）該形質転換体により生産されたヘキソサミンを分離する工程、
を含むヘキソサミンの製造方法。
１５． GFAT遺伝子が、クロレラウイルス由来ＧＦＡＴ遺伝子であることを特徴とする項１４に記載のヘキソサミンの製造方法。
１６． GFAT遺伝子が、配列番号2記載の遺伝子であることを特徴とする項１４又は１５
に記載のヘキソサミンの製造方法。
１７． GFAT遺伝子が、配列番号2記載の遺伝子の一部、または欠失体、または付加体、
または置換体であって、かつＧＦＡＴ活性を有するポリペプチドをコードする遺伝子であることを特徴とする項１４又は１５に記載のヘキソサミンの製造方法。
１８． 形質転換体を生育する工程が、該形質転換体を培養する工程であることを特徴とする項１４に記載のヘキソサミンの製造方法。
１９． ヘキソサミンが、Ｎ−アセチルグルコサミン、またはＮ−アセチルグルコサミン−６−リン酸であることを特徴とする項１〜９のいずれかに記載のヘキソサミン高生産形質転換植物細胞又は形質転換植物又はその子孫又はそれらの器官又はそれらの組織。
２０． ヘキソサミンが、Ｎ−アセチルグルコサミン、またはＮ−アセチルグルコサミン−６−リン酸であることを特徴とする項１０記載の発現用組換えベクター。
２１． ヘキソサミンが、Ｎ−アセチルグルコサミン、またはＮ−アセチルグルコサミン−６−リン酸であることを特徴とする項１３記載の植物抽出物。
２２． ヘキソサミンが、Ｎ−アセチルグルコサミン、またはＮ−アセチルグルコサミン−６−リン酸であることを特徴とする項１４〜１８のいずれかに記載のヘキソサミンの製造方法。
That is, the present invention relates to a hexosamine high-producing transformed plant cell or transformed plant or its progeny or organ or tissue thereof, a recombinant vector for expression, a plant extract, and a method for producing hexosamine.
1. A hexosamine-producing high-transformed plant cell or transformed plant, or its progeny or organ or tissue thereof, wherein an exogenous GFAT (glutamine: fructose-6-phosphate amide transferase) gene is introduced.
2.
3.
4).
5. A hexosamine high-producing transformed plant cell or transformed plant, or a progeny thereof, an organ thereof, or a tissue thereof, wherein a chlorella virus-derived GFAT gene is introduced.
6). A hexosamine high-producing transformed plant cell or transformed plant, or a progeny thereof, an organ thereof or a tissue thereof, wherein the chlorella virus-derived GFAT gene described in SEQ ID NO: 2 is introduced.
7). Item 7. The hexosamine high-production transformed plant according to any one of
8). Item 7. The hexosamine high-producing trait according to any one of
9. Item 7. The hexosamine high-production transformed plant according to any one of
10. A vector for obtaining a hexosamine high-producing transformed plant or a progeny having the same properties, or an organ thereof or a tissue thereof, wherein a chlorella virus-derived GFAT gene is introduced into an expression recombinant vector A recombinant vector for expression.
11. Item 11. The expression recombinant vector according to Item 10, wherein the chlorella virus-derived GFAT gene is the GFAT gene described in SEQ ID NO: 2.
12 Item 10. A plant extract obtained from the plant according to any one of
13. Item 13. The plant extract according to Item 12, wherein the plant extract is hexosamine.
14 (1) a step of transforming a plant cell using a recombinant vector for expression, wherein an exogenous GFAT (glutamine: fructose-6-phosphate amide transferase) gene is introduced;
(2) a step of growing a transformant (transformed plant cell or transformed plant) obtained by transformation,
(3) a step of separating hexosamine produced by the transformant,
A method for producing hexosamine, comprising:
15. Item 15. The method for producing hexosamine according to Item 14, wherein the GFAT gene is a chlorella virus-derived GFAT gene.
16. Item 14 or 15 wherein the GFAT gene is the gene set forth in SEQ ID NO: 2.
A method for producing hexosamine as described in 1. above.
17. GFAT gene is a part of the gene shown in SEQ ID NO: 2, or a deletion or adduct,
Item 16. The method for producing hexosamine according to Item 14 or 15, wherein the gene is a substitute and a gene encoding a polypeptide having GFAT activity.
18. Item 15. The method for producing hexosamine according to Item 14, wherein the step of growing the transformant is a step of culturing the transformant.
19. Item 10. The hexosamine high-producing transformed plant cell or transformed plant or its progeny or progeny thereof according to any one of
20. Item 11. The recombinant expression vector according to Item 10, wherein the hexosamine is N-acetylglucosamine or N-acetylglucosamine-6-phosphate.
21. Item 14. The plant extract according to Item 13, wherein the hexosamine is N-acetylglucosamine or N-acetylglucosamine-6-phosphate.
22. Item 18. The method for producing hexosamine according to any one of Items 14 to 18, wherein the hexosamine is N-acetylglucosamine or N-acetylglucosamine-6-phosphate.
本発明によると、植物細胞または植物体から有用なＧｌｃＮＡｃ、GlcNAc-6-Pなどのヘキソサミンを安全、安価、低環境負荷に生産することができる。特にＧｌｃＮＡｃは、甘味料としての用途だけではなく、変形関節炎の治療を目的とした医薬用途にも利用されており、本発明により食品分野、医薬分野の研究開発を活性化でき、産業界に大きく寄与することができる。 According to the present invention, hexosamine such as GlcNAc and GlcNAc-6-P useful from plant cells or plants can be produced safely, inexpensively and with a low environmental load. In particular, GlcNAc is used not only as a sweetener but also for pharmaceutical use for the treatment of osteoarthritis. The present invention can activate research and development in the food and pharmaceutical fields, and is greatly applied to the industry. Can contribute.
本発明においては、ＧＦＡＴをコードするDNA又はＧＦＡＴの一部又はＧＦＡＴの欠失、付加又は置換体であって且つＧＦＡＴ活性を有するポリペプチドをコードするDNAを用いて、植物細胞又は植物体の形質転換を行う。
GFAT遺伝子を植物体に導入する際には、形質転換する植物と由来が異なるもの（即ち、外因性のGFAT遺伝子）が望ましい。例えば、タバコに由来の異なるクロレラウイルス由来GFATまたは大腸菌由来GFATを導入することが例示されるが、これに限定されるものではない。また、由来を同じとするGFAT遺伝子の使用はサイレンシングの点から好ましくないが、一方、サイレンシングが起こらないようにGFAT遺伝子を改良し、由来の同じ植物に導入することは、本発明の効果を損なわない範囲で使用してもよい。従って、本発明の外因性GFAT遺伝子は、サイレンシングが起こらない程度に改変したGFAT遺伝子変異体を包含する。
In the present invention, using a DNA encoding GFAT or a part of GFAT or a deletion, addition or substitution of GFAT and a polypeptide having GFAT activity, Make a conversion.
When the GFAT gene is introduced into a plant body, it is desirable that the origin is different from the plant to be transformed (that is, exogenous GFAT gene). For example, introduction of chlorate virus-derived GFAT or Escherichia coli-derived GFAT derived from different tobacco is exemplified, but the present invention is not limited thereto. In addition, the use of the GFAT gene having the same origin is not preferable from the viewpoint of silencing. On the other hand, it is advantageous to improve the GFAT gene so that silencing does not occur and introduce it into the same plant of origin. You may use in the range which does not impair. Therefore, the exogenous GFAT gene of the present invention includes a GFAT gene variant modified to such an extent that silencing does not occur.
ＧＦＡＴとしては、Ｌ−グルタミンとフルクトース−６−リン酸を基質として、グルコサミン−６−リン酸を合成するものであれば、その由来は特に限定されない。例えば、真核生物由来、原核生物由来、ウイルス由来があげられる。真核生物由来としてはヒト、マウス、トウモロコシ、線虫、酵母、原核生物由来としては枯草菌、大腸菌、ウイルス由来としてはクロレラウイルスのＧＦＡＴを用いることができるが、これに限定されるものではない。 The origin of GFAT is not particularly limited as long as it synthesizes glucosamine-6-phosphate using L-glutamine and fructose-6-phosphate as substrates. Examples include eukaryotic origin, prokaryotic origin, and virus origin. Examples of eukaryotic origin include human, mouse, corn, nematode, yeast, prokaryotic origin, Bacillus subtilis, Escherichia coli, and viral origin, chlorella virus GFAT, but is not limited thereto. .
ＧＦＡＴ遺伝子には、ＧＦＡＴ1、ＧＦＡＴ２等の各種タイプを有するものがあるが、種類は特に限定されない。また、GFAT遺伝子としては、形質転換する植物の内在性GFAT遺伝子と相同性の低いものが好ましい。形質転換する植物の内在性GFAT遺伝子と相同性の低い組み合わせのGFAT遺伝子としては、動物由来GFAT遺伝子、菌由来GFAT遺伝子、原核生物由来GFAT遺伝子、ウイルス由来GFAT遺伝子がある。これらのGFAT遺伝子でウイルス由来GFAT遺伝子が好ましい。さらに好ましくは、これらのウイルス由来GFAT遺伝子の中でも、クロレラウイルス由来のＧＦＡＴがよい。特に好ましくは配列番号2の遺伝子がよい。クロレラウイルス由来グルタミン：フルクトース−６−リン酸 アミドトランスフェラーゼ遺伝子（cvGFAT）を得るためには、クロレラウイルスPBCV1系統の配列情報などを参考にPCRプライマーを作製すればよい。好ましくは、GFAT推定領域の外側から増幅できるように、プライマーを設計し、クロレラウイルスのゲノムDNAを鋳型としてＰＣＲで得られた遺伝子断片をpCRscript（Stratagene製）などにクローニングすれば良い。 Some GFAT genes have various types such as GFAT1 and GFAT2, but the type is not particularly limited. Further, as the GFAT gene, those having low homology with the endogenous GFAT gene of the plant to be transformed are preferable. Examples of GFAT genes that have a low homology with the endogenous GFAT gene of the plant to be transformed include animal-derived GFAT genes, fungal-derived GFAT genes, prokaryotic-derived GFAT genes, and virus-derived GFAT genes. Of these GFAT genes, virus-derived GFAT genes are preferred. More preferably, among these virus-derived GFAT genes, chlorella virus-derived GFAT is preferable. The gene of SEQ ID NO: 2 is particularly preferable. In order to obtain a chlorella virus-derived glutamine: fructose-6-phosphate amide transferase gene (cvGFAT), a PCR primer may be prepared with reference to the sequence information of the chlorella virus PBCV1 strain. Preferably, a primer is designed so that it can be amplified from outside the GFAT estimation region, and a gene fragment obtained by PCR using chlorella virus genomic DNA as a template is cloned into pCRscript (Stratagene).
配列番号２のクロレラウイルス弘前系統(CVHI1)由来GFAT遺伝子は上記方法により得られ、CVHI1株由来GFAT遺伝子cvGFAT-HIのDNA配列を決定した。かくして、塩基配列決定によって新規な配列の遺伝子を得、これを「cvGFAT-HI遺伝子」と命名した。得られた遺伝子は、そのコード領域の全長が配列番号２で示される１７８８塩基からなり、該塩基配列によってコードされるアミノ酸配列は、配列番号１に示される５９５個のアミノ酸配列からなっていた。該アミノ酸配列は、公知のクロレラウイルスPBCV-1系統、K2系統とアミノ酸レベルで98％の相同性を示した。 The GFAT gene derived from the chlorella virus Hirosaki strain (CVHI1) of SEQ ID NO: 2 was obtained by the method described above, and the DNA sequence of the GFAT gene cvGFAT-HI derived from the CVHI1 strain was determined. Thus, a gene having a novel sequence was obtained by nucleotide sequencing, and this was named “cvGFAT-HI gene”. The obtained gene consisted of 1788 bases whose full length of the coding region is represented by SEQ ID NO: 2, and the amino acid sequence encoded by the base sequence consisted of 595 amino acid sequences represented by SEQ ID NO: 1. The amino acid sequence showed 98% homology with the known chlorella virus PBCV-1 and K2 strains at the amino acid level.
ＧＦＡＴの一部であって、且つＧＦＡＴ活性を有するポリペプチドとは、上記のようなＧＦＡＴにおいてＧＦＡＴ活性を奏するために必須となるアミノ酸部位を含み、かつＧＦＡＴ活性を有するポリペプチドである。 The polypeptide that is a part of GFAT and has GFAT activity is a polypeptide that includes an amino acid site that is essential for exhibiting GFAT activity in GFAT as described above and has GFAT activity.
ＧＦＡＴの欠失、付加又は置換体であって、且つＧＦＡＴ活性を有するポリペプチドとは、上記のようなＧＦＡＴのアミノ酸配列において、１若しくは複数個、好ましくは１も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により表され、且つ、ＧＦＡＴ活性を失わない程度の変異がなされたポリペプチドである。 A polypeptide having a deletion, addition or substitution of GFAT and having GFAT activity is a deletion of one or more, preferably one or several amino acids in the amino acid sequence of GFAT as described above, A polypeptide represented by a substituted or added amino acid sequence and mutated to such an extent that GFAT activity is not lost.
このような変異は、自然界において生じるほかに、人為的な変異も含む。変異したアミノ酸の数は、ＧＦＡＴ活性を失わない限り、その個数は特に制限されない。天然の変異例として、配列番号１のクロレラウイルス弘前系統のＧＦＡＴと、公知のクロレラウイルスPBCV-1系統、K2系統のＧＦＡＴとは、少なくともアミノ酸レベルで２％が変異している例がある。 Such mutations occur in nature as well as artificial mutations. The number of mutated amino acids is not particularly limited as long as GFAT activity is not lost. As an example of natural mutation, there is an example in which at least 2% of GFAT of the chlorella virus Hirosaki strain of SEQ ID NO: 1 and GFAT of the known chlorella virus PBCV-1 strain and K2 strain are mutated at the amino acid level.
本発明におけるＧＦＡＴをコードするDNA又はＧＦＡＴの一部又はＧＦＡＴの欠失、付加又は置換体であって且つＧＦＡＴ活性を有するポリペプチドをコードするDNAとしては、上記ＧＦＡＴ又はポリペプチドをコードするものであれば特に限定されず、コドンの縮重により配列が異なるものも含まれる。 In the present invention, DNA encoding GFAT or polypeptide is a part of GFAT or a part of GFAT or a deletion, addition or substitution of GFAT and encoding a polypeptide having GFAT activity. There is no particular limitation as long as it is present, and it includes those having different sequences due to degeneracy of codons.
上記（ｉ）ＧＦＡＴをコードするDNA又は（ｉｉ）ＧＦＡＴの一部又はＧＦＡＴの欠失、付加又は置換体であって且つＧＦＡＴ活性を有するポリペプチドをコードするDNAを含む発現用組換えベクターを用いて植物細胞又は植物体を形質転換することによって、本発明の形質転換体、換言すると、形質転換植物細胞又は形質転換植物が作製される。 Using the above-mentioned (i) DNA encoding GFAT or (ii) a recombinant vector for expression containing a DNA encoding a polypeptide having GFAT activity, which is a part of GFAT or a deletion, addition or substitution of GFAT By transforming the plant cell or the plant body, the transformant of the present invention, in other words, the transformed plant cell or the transformed plant is produced.
本発明の形質転換植物細胞又は形質転換植物は、（ｉ）ＧＦＡＴをコードするDNA又は（ｉｉ）ＧＦＡＴの一部又はＧＦＡＴの欠失、付加又は置換体であって且つＧＦＡＴ活性を有するポリペプチドをコードするDNAを挿入した発現用組換えベクターを、目的遺伝子が発現し得るように宿主中に導入し、形質転換することにより得ることができる。 The transformed plant cell or transformed plant of the present invention comprises (i) a DNA encoding GFAT or (ii) a part of GFAT, a deletion, addition or substitution of GFAT, and a polypeptide having GFAT activity. A recombinant expression vector into which the DNA to be encoded is inserted can be obtained by introducing it into a host and transforming it so that the target gene can be expressed.
ここで、宿主とは、植物全体、種子、植物器官(例えば葉、花弁、茎、根、根茎等)、植物組織(例えば表皮、師部、柔組織、木部、維管束等)又は植物培養細胞のいずれをも意味するものである。 Here, the host refers to the whole plant, seed, plant organ (for example, leaf, petal, stem, root, rhizome, etc.), plant tissue (for example, epidermis, phloem, soft tissue, xylem, vascular bundle, etc.) or plant culture. Any cell is meant.
本明細書において、植物とは、種子植物、シダ植物、コケ植物、地衣植物等を含む、多細胞の植物を意味し、植物全体、種子、植物器官(例えば葉、花弁、茎、根、根茎等)、植物組織(例えば表皮、師部、柔組織、木部、維管束等)又は植物培養細胞のいずれをも包含するものである。 In the present specification, the plant means a multicellular plant including a seed plant, a fern plant, a moss plant, a lichen plant, etc., and the whole plant, seed, plant organ (for example, leaf, petal, stem, root, rhizome) Etc.), plant tissue (for example, epidermis, phloem, soft tissue, xylem, vascular bundle, etc.) or plant cultured cells.
また、該形質転換して得られた形質転換体（形質転換植物細胞又は形質転換植物）を培養し、該形質転換体により生産されたヘキソサミンを分離することにより、ヘキソサミンが製造される。 Further, hexosamine is produced by culturing a transformant (transformed plant cell or transformed plant) obtained by the transformation and separating hexosamine produced by the transformant.
発現用組換えベクターとしては、形質転換植物細胞又は形質転換植物作製のために通常用いられているベクターを用いることができる。 As a recombinant vector for expression, a vector usually used for producing a transformed plant cell or a transformed plant can be used.
このようなベクターとしては、植物細胞で転写可能なプロモーター配列と転写産物の安定化に必要なポリアデニレーション部位を含むターミネーター配列を含んでいれば特に制限はない。例えばプラスミド「pBI121」、「pBI221」、「pBI101」、「pIG121Hm」等を用いることができる。 Such a vector is not particularly limited as long as it contains a promoter sequence that can be transcribed in plant cells and a terminator sequence including a polyadenylation site necessary for stabilization of the transcription product. For example, plasmids “pBI121”, “pBI221”, “pBI101”, “pIG121Hm” and the like can be used.
植物培養細胞を宿主として用いる場合は、形質転換は、植物培養細胞に、エレクトロポレーション法又はアグロバクテリウムのバイナリーベクター法もしくはパーティクルガン法で（ｉ）ＧＦＡＴをコードするDNA又は（ｉｉ）ＧＦＡＴの一部又はＧＦＡＴの欠失、付加又は置換体であって且つＧＦＡＴ活性を有するポリペプチドをコードするDNAを挿入した発現用組換えベクターを導入することにより行うことができる。発現用組換えベクターを導入された植物細胞は、例えば、カナマイシン耐性等の薬剤耐性を基準として選択される。形質転換された植物細胞は、細胞培養、組織培養、器官培養に用いることができ、また従来知られている植物組織培養法等を用いて、植物体を再生することもできる。 When plant cultured cells are used as a host, transformation is performed on plant cultured cells by electroporation, Agrobacterium binary vector method or particle gun method (i) DNA encoding GFAT or (ii) GFAT It can be carried out by introducing a recombinant vector for expression into which a DNA encoding a polypeptide which is a partial, GFAT deletion, addition or substitution product and has GFAT activity is inserted. Plant cells introduced with the expression recombinant vector are selected based on drug resistance such as kanamycin resistance, for example. The transformed plant cell can be used for cell culture, tissue culture and organ culture, and the plant body can also be regenerated using a conventionally known plant tissue culture method or the like.
形質転換の対象となる植物細胞の例としては、例えば、タバコ由来BY-2細胞やT-13細胞、ニンジン由来kurodagosun細胞、ブドウ由来VR細胞やVW細胞、ヨウシュヤマゴボウ由来PAR細胞やPAP細胞やPAW細胞、シロイヌナズナ由来T87細胞、アスパラガス由来Asp-86細胞やA.per細胞やA.pas細胞やA.plo細胞、スイカ由来Cba-1細胞、トマト由来Sly-1細胞、ハッカ由来1-Mar細胞、ニチニチソウ由来CRA細胞やV208細胞や、ホウレンソウ由来Spi-WT細胞やSpi-I-1細胞やSpi-12F細胞、ヘチマ由来Lcy-1細胞やLcyD6細胞やLcyD7細胞や、イネ由来OS-1細胞、ツルニチニチソウ由来Vma-1細胞、ゴマ由来PSB細胞やPSW細胞やPSG細胞、ヒャクニチソウ由来ZE3細胞等が挙げられる。 Examples of plant cells to be transformed include, for example, tobacco-derived BY-2 cells and T-13 cells, carrot-derived kurodagosun cells, grape-derived VR cells and VW cells, pokeweed-derived PAR cells and PAP cells, PAW cells, Arabidopsis-derived T87 cells, Asparagus-derived Asp-86 cells, A.per cells, A.pas cells, A.plo cells, watermelon-derived Cba-1 cells, tomato-derived Sly-1 cells, mint-derived 1-Mar Cells, periwinkle-derived CRA cells and V208 cells, spinach-derived Spi-WT cells, Spi-I-1 and Spi-12F cells, loofah-derived Lcy-1 cells, LcyD6 and LcyD7 cells, and rice-derived OS-1 cells , Periwinkle-derived Vma-1 cells, sesame-derived PSB cells, PSW cells, PSG cells, zinnia-derived ZE3 cells, and the like.
植物体、植物器官又は植物組織を宿主とする場合、形質転換は、採取した植物切片に、アグロバクテリウムのバイナリーベクター法又はパーティクルボンバードメント法によって、あるいはプロトプラストにエレクトロポレーション法によって、（ｉ）ＧＦＡＴをコードするDNA又は（ｉｉ）ＧＦＡＴの一部又はＧＦＡＴの欠失、付加又は置換体であって且つＧＦＡＴ活性を有するポリペプチドをコードするDNAを挿入した発現用組換えベクターを導入し、形質転換の結果得られる腫瘍組織、カルスやシュート、毛状根等を分離することにより行われる。 In the case of using a plant body, plant organ or plant tissue as a host, transformation is performed on the collected plant section by the Agrobacterium binary vector method or particle bombardment method, or by protoplast electroporation method (i) Introducing a recombinant vector for expression into which a DNA encoding GFAT or (ii) a part of GFAT or a deletion, addition or substitution of GFAT and a DNA encoding a polypeptide having GFAT activity is inserted, It is performed by separating tumor tissue, callus, shoots, hairy roots, and the like obtained as a result of conversion.
こうして得られる腫瘍組織、カルスやシュート、毛状根等は、そのまま細胞培養、組織培養又は器官培養に用いることが可能である。また従来知られている植物組織培養法を用い、適当な濃度の植物ホルモンの投与等により植物体に再生させることができる。 The tumor tissue, callus, shoot, hairy root and the like thus obtained can be used as they are for cell culture, tissue culture or organ culture. Further, it can be regenerated into a plant body by administration of an appropriate concentration of a plant hormone using a conventionally known plant tissue culture method.
ＧＦＡＴ遺伝子が導入された植物細胞から植物を再生させるには、このような植物細胞を、再分化培地、ホルモンフリーのムラシゲ・スクーグ（ＭＳ）培地等に培養すればよい。発根した幼植物体は、土壌に移植して栽培することにより植物体とすることができる。再生（再分化）の方法は植物細胞の種類により異なるが、従来知られている植物組織培養法を適宜用いることができる。 In order to regenerate a plant from a plant cell into which a GFAT gene has been introduced, such a plant cell may be cultured in a regeneration medium, a hormone-free Murashige-Skoog (MS) medium, or the like. The rooted young plant body can be made into a plant body by transplanting and cultivating it in soil. Although the method of regeneration (redifferentiation) varies depending on the type of plant cell, a conventionally known plant tissue culture method can be appropriately used.
例えばイネではFujimuraら（Fujimuraら（１９９５）、Plant Tissue Culture Lett.、vol.２：ｐ７４）の方法を用いることができる。トウモロコシでは、Shillitoら（Shillitoら（１９８９）、Bio/Technology、vol.7:p581、Ｇｏｒｄｅｎ−Ｋａｍｍ，１９９０，Ｐｌａｎｔ Ｃｅｌｌ ２，６０３）の方法を用いることができる。ジャガイモでは、Visserら（Visserら（１９８９）、Theor.Appl.Genet.、vol.78:p589）の方法を用いることができる。タバコでは、Nagataら（Ｎａｇａｔａ，１９７１，Ｐｌａｎｔａ ９９，１２）の方法を用いることができる。シロイヌナズナではAkamaら（Akamaら（１９９２）、Plant Cell Rep., vol.12:p7）の方法を用いることができる。 For example, in rice, the method of Fujimura et al. (Fujimura et al. (1995), Plant Tissue Culture Lett., Vol. 2: p74) can be used. In maize, the method of Shillito et al. (Shillito et al. (1989), Bio / Technology, vol. 7: p581, Gorden-Kamm, 1990, Plant Cell 2,603) can be used. In potato, the method of Visser et al. (Visser et al. (1989), Theor. Appl. Genet., Vol. 78: p589) can be used. For tobacco, the method of Nagata et al. (Nagata, 1971, Planta 99, 12) can be used. In Arabidopsis, the method of Akama et al. (Akama et al. (1992), Plant Cell Rep., Vol. 12: p7) can be used.
これらの方法により作製された植物体、または同じ性質を有するその子孫（繁殖媒体、例えば種子、塊茎、切穂等から得た植物体）も本発明の対象である。 Plants produced by these methods, or their progeny having the same properties (plants obtained from propagation media such as seeds, tubers, cut ears, etc.) are also objects of the present invention.
植物内で、ＧＦＡＴ活性を持つ酵素を発現させ、更に植物内でヘキソサミンを生産、蓄積または分泌させる際には、GFATを定常発現プロモーターを用いて植物全体で発現させても良く、植物の適切な組織および器官で特異的に発現するようにＧＦＡＴ遺伝子を制御することが好ましい。 When an enzyme having GFAT activity is expressed in a plant and hexosamine is produced, accumulated or secreted in the plant, GFAT may be expressed in the whole plant using a constant expression promoter. It is preferred to control the GFAT gene so that it is specifically expressed in tissues and organs.
そのような制御を行うためには組織特異的又は器官特異的プロモーターを更に発現用組換えベクターに挿入して用いるとよい。 In order to perform such control, a tissue-specific or organ-specific promoter may be further inserted into the expression recombinant vector.
また、時期特異的プロモーターを用いれば、特定の時期だけに目的遺伝子を発現させることができ、特定の時期だけに生産性を改良することができる。例えば、栄養生長期に働くプロモーターを用いることによって、栄養生長期のみで生産性を改良することができる。 In addition, when a time-specific promoter is used, a target gene can be expressed only at a specific time, and productivity can be improved only at a specific time. For example, by using a promoter that works in the vegetative growth period, productivity can be improved only in the vegetative growth period.
器官特異的プロモーターとしては、例えば、根特異的プロモーター、塊茎特異的プロモーター、葉特異的プロモーター、種子特異的プロモーター、茎特異的プロモーター等がある。 Examples of the organ-specific promoter include a root-specific promoter, a tuber-specific promoter, a leaf-specific promoter, a seed-specific promoter, and a stem-specific promoter.
また、組織特異的プロモーターとしては、例えば、緑色組織特異的プロモーター等がある。 Examples of the tissue-specific promoter include a green tissue-specific promoter.
より具体的に、使用し得るプロモーターの例としては、例えば、構成的高発現プロモーターとして、カリフラワーモザイクウイルスの35ＳRNA遺伝子のプロモーターであるCaMV35Sプロモーター等が挙げられる。緑色組織特異的プロモーターとしては、リブロース1,5-ビスリン酸カルボキシラーゼの小サブユニットタンパク質をコードするrbs遺伝子のプロモーターやクロロフィルa/b結合タンパク質をコードするCAB遺伝子のプロモーター、グルセルアルデヒド3-リン酸デヒドロゲナーゼAサブユニットタンパク質をコードするGapA遺伝子プロモーター等が挙げられる。また種子特異的プロモーターとしては、リポキシゲナーゼ遺伝子のLOXプロモーター、レクチン遺伝子のPslプロモーター、アミラーゼ遺伝子のAmylAプロモーター等が挙げられる。根特異的プロモーターとしては、ヒヨシアミン6b-ヒドロキラーゼ遺伝子のA6H6Hプロモーター、プトレシンN-メチルトランスフェラーゼのPMTプロモーター等が挙げられる。茎特異的プロモーターとしては、スクロースシンターゼのSus4遺伝子プロモーター、グリコプロテインをコードするパタチン遺伝子プロモーター等が挙げられる。
More specifically, examples of promoters that can be used include, for example, the CaMV35S promoter, which is a promoter of the 35S RNA gene of cauliflower mosaic virus, as a constitutive high expression promoter. Green tissue-specific promoters include the rbs gene promoter encoding the small subunit protein of
また、ＧＦＡＴ遺伝子の発現を誘導性プロモーターで制御することも考えられる。誘導性プロモーターの例を以下に記載する。 It is also conceivable to control the expression of the GFAT gene with an inducible promoter. Examples of inducible promoters are described below.
傷害やサリチル酸の添加により発現が増加する耐病性関連遺伝子プロモーターであるPR1aプロモーターや、乾燥、低温、高塩濃度、アブシジン酸の転換より発現が増加するrd29A遺伝子プロモーター等が挙げられる。農薬として用いられている化合物により発現が誘導されるプロモーターとしては、除草剤のセーフナーにより誘導されるグルタチオン-S-トランスフェラーゼの27KDaサブユニットタンパク質をコードするGST-27遺伝子プロモーター、ベンゾ（1,2,3）-チアジアゾール-７-カルボシオイック酸Ｓ−メチルエステル（BTH）により誘導されるキチナーゼ遺伝子プロモーターやPR遺伝子タンパク質プロモーター等がある。さらに、植物細胞内でＧＦＡＴ遺伝子をより安定に発現させるためにインスレーターの利用や目的の細胞内小器官でＧＦＡＴを局在させるためにシグナルペプチドを付加したり、ＧＦＡＴの一部を置換および欠損させること等を行ってもよい。 Examples include the PR1a promoter which is a disease resistance-related gene promoter whose expression is increased by the addition of injury or salicylic acid, and the rd29A gene promoter whose expression is increased by drought, low temperature, high salt concentration, or abscisic acid conversion. Promoters whose expression is induced by compounds used as agricultural chemicals include the GST-27 gene promoter encoding the 27 KDa subunit protein of glutathione-S-transferase induced by the herbicide safener, benzo (1,2, 3) There are chitinase gene promoter, PR gene protein promoter and the like induced by -thiadiazole-7-carbocioic acid S-methyl ester (BTH). In addition, use of an insulator for more stable expression of the GFAT gene in plant cells, addition of a signal peptide to localize GFAT in the target organelle, substitution or deletion of a part of GFAT It may be performed.
形質転換の対象となる植物体には、遺伝子導入の可能ないずれの植物も包含される。 Plants to be transformed include any plant capable of gene transfer.
本発明の植物又は植物体には、被子植物の単子葉植物や双子葉植物、裸子植物等が包含される。このような植物には、任意の有用植物、特に作物植物、蔬菜植物、花卉植物や木本植物が含まれる。 The plant or plant of the present invention includes angiosperm monocotyledons, dicotyledons, gymnosperms and the like. Such plants include any useful plant, especially crop plants, sugar beet plants, flower plants and woody plants.
また、本発明の植物又は植物体には、シダ植物、コケ植物等も含まれる。 The plant or plant of the present invention includes fern plants and moss plants.
本発明が使用され得る植物種の例としては、具体的には、ナス科、イネ科、アブラナ科、バラ科、マメ科、ウリ科、シソ科、ユリ科、アカザ科、セリ科、フトモト科、ヒルガオ科の植物等が挙げられる。 Specific examples of the plant species in which the present invention can be used include, And plants of the convolvulaceae family.
ナス科の植物の例としては、Ｎｉｃｏｔｉａｎａ、Ｓｏｌａｎｕｍ、Ｄａｔｕｒａ、Ｌｙｃｏｐｅｒｓｉｏｎ、またはＰｅｔｕｎｉａに属する植物が挙げられ、例えば、タバコ、ナス、ジャガイモ、トマト、トウガラシ、ペチュニア等が含まれる。 Examples of solanaceous plants include plants belonging to Nicotiana, Solanum, Dataura, Lycopersion, or Petunia, and include tobacco, eggplant, potato, tomato, pepper, petunia, and the like.
イネ科の植物の例としては、Ｏｒｙｚａ、Ｈｏｒｄｅｎｕｍ、Ｓｅｃａｌｅ、Ｓｃｃｃｈａｒｕｍ、Ｅｃｈｉｎｏｃｈｌｏａ、Ｚｅａ、またはＳａｃｃｈａｒｕｍに属する植物が挙げられ、例えば、イネ、オオムギ、ライムギ、ヒエ、モロコシ、トウモロコシ、サトウキビ等が含まれる。 Examples of Gramineae plants include plants belonging to Oryza, Hordenum, Secale, Scccharum, Echinochloa, Zea, or Saccharum, including, for example, rice, barley, rye, barnyard millet, sorghum, corn, and sugarcane.
アブラナ科の植物の例としては、Ｒａｐｈａｎｕｓ、Ｂｒａｓｓｉｃａ、Ａｒａｂｉｄｏｐｓｉｓ、Ｗａｓａｂｉａ、またはＣａｐｓｅｌｌａに属する植物が挙げられ、例えば、大根、アブラナ、シロイヌナズナ、ワサビ、ナズナ等が含まれる。 Examples of cruciferous plants include plants belonging to Raphanus, Brassica, Arabidopsis, Wasabia, or Capsella, and include, for example, radish, rape, Arabidopsis thaliana, horseradish, tuna and the like.
バラ科の植物の例としては、Ｏｒｕｎｕｓ、Ｍａｌｕｓ、Ｐｙｎｕｓ、Ｆｒａｇａｒｉａ、またはＲｏｓａに属する植物が挙げられ、例えば、ウメ、モモ、リンゴ、ナシ、オランダイチゴ、バラ等が含まれる。 Examples of Rosaceae plants include plants belonging to Orrunus, Malus, Pynus, Fragaria, or Rosa, and examples include ume, peach, apple, pear, Dutch strawberry, rose and the like.
マメ科の植物の例としては、Ｇｌｙｃｉｎｅ、Ｖｉｇｎａ、Ｐｈａｓｅｏｌｕｓ、Ｐｉｓｕｍ、Ｖｉｃｉａ、Ａｒａｃｈｉｓ、Ｔｒｉｆｏｌｉｕｍ、Ａｌｐｈａｌｆａ、またはＭｅｄｉｃａｇｏに属する植物が挙げられ、例えば、ダイズ、アズキ、インゲンマメ、エンドウ、ソラマメ、ラッカセイ、クローバ、ウマゴヤシ等が含まれる。 Examples of leguminous plants include plants belonging to Glycine, Vigna, Phaseolus, Pisum, Via, Arachis, Trifolium, Alphafa, or Medicago, such as soybean, azuki bean, kidney bean, pea, broad bean, peanut, peanut, peanut, Includes marinated coconut.
ウリ科の植物の例としては、Ｌｕｆｆａ、Ｃｕｃｕｒｂｉｔａ、またはＣｕｃｕｍｉｓに属する植物が挙げられ、例えば、ヘチマ、カボチャ、キュウリ、メロン等が含まれる。 Examples of cucurbitaceae plants include plants belonging to Luffa, Cucurbita, or Cucumis, and include, for example, loofah, pumpkin, cucumber, melon and the like.
シソ科の植物の例としては、Ｌａｖａｎｄｕｌａ、Ｍｅｎｔｈａ、またはＰｅｒｉｌｌａに属する植物が挙げられ、例えば、ラベンダー、ハッカ、シソ等が含まれる。 Examples of Labiatae plants include plants belonging to Lavandula, Mentha, or Perilla, and include, for example, lavender, mint, perilla and the like.
ユリ科に属する植物の例としては、Ａｌｌｉｕｍ、Ｌｉｌｉｕｍ、またはＴｕｌｉｐａに属する植物が挙げられ、例えば、ネギ、ニンニク、ユリ、チューリップ等が含まれる。 Examples of plants belonging to the lily family include plants belonging to Allium, Lilium, or Tulipa, and include, for example, leeks, garlic, lilies, tulips and the like.
アカザ科の植物の例としては、Ｓｐｉｎａｃｉａに属する植物が挙げられ、例えば、テンサイ、ホウレンソウ等が含まれる。 Examples of the plant of the family Rabbitaceae include plants belonging to Spinacaia, and include, for example, sugar beet, spinach and the like.
セリ科の植物の例としては、Ａｎｇｅｌｉｃａ、Ｄａｕｃｕｓ、Ｃｒｙｐｔｏｔａｅｎｉａ、またはＡｐｉｔｕｍに属する植物が挙げられ、例えば、シシウド、ニンジン、ミツバ、セロリ等が含まれる。 Examples of celery family plants include plants belonging to Angelica, Daucus, Cryptotaenia, or Apitum, and include, for example, shishiudo, carrot, honeybee, celery and the like.
ヒルガオ科の植物の例としては、Ｉｐｏｍｏｅａに属する植物が挙げられ、例えば、サツマイモ等が含まれる。 Examples of the convolvulaceae plant include plants belonging to Ipomoea, including, for example, sweet potato.
上記のような形質転換植物と同じ性質を有する子孫、また、それらの器官及び組織も本発明の対象である。 Progeny having the same properties as the transformed plant as described above, and their organs and tissues are also objects of the present invention.
また、本発明には、ＧＦＡＴを生産する形質転換植物細胞が含まれる。また、ＧＦＡＴを生産する形質転換植物又はそれと同じ性質を有する子孫又はそれらの器官又はそれらの組織も含まれる。 The present invention also includes transformed plant cells that produce GFAT. Also included are transformed plants that produce GFAT or progeny having the same properties or organs or tissues thereof.
ＧＦＡＴを生産する形質転換植物細胞は、（ｉ）ＧＦＡＴをコードするDNA又は（ｉｉ）ＧＦＡＴの一部又はＧＦＡＴの欠失、付加又は置換体であって且つＧＦＡＴ活性を有するポリペプチドをコードするDNAを含む発現用組換えベクターを用いて植物細胞を形質転換することにより得ることができる。 Transformed plant cells that produce GFAT are: (i) DNA encoding GFAT or (ii) DNA encoding a polypeptide that is a part of GFAT or a deletion, addition or substitution of GFAT and has GFAT activity It can be obtained by transforming a plant cell using a recombinant vector for expression containing
ＧＦＡＴを生産する形質転換植物は、ＧＦＡＴをコードするDNA又は（ｉｉ）ＧＦＡＴの一部又はＧＦＡＴの欠失、付加又は置換体であって且つＧＦＡＴ活性を有するポリペプチドをコードするDNAを含む発現用組換えベクターを用いて植物細胞を形質転換し、該形質転換細胞からヘキソサミンを高生産する植物体を再生することにより得ることができる。 A transformed plant producing GFAT is for expression comprising DNA encoding GFAT or (ii) a part of GFAT or a deletion, addition or substitution of GFAT and a DNA encoding a polypeptide having GFAT activity It can be obtained by transforming a plant cell using a recombinant vector and regenerating a plant body that highly produces hexosamine from the transformed cell.
該形質転換植物細胞又は植物内においては、ＧＦＡＴ遺伝子がＧＦＡＴ活性を持つ酵素として発現される。 In the transformed plant cell or plant, the GFAT gene is expressed as an enzyme having GFAT activity.
上記のようなＧＦＡＴを生産する形質転換植物細胞或いは形質転換植物を用いることにより、ヘキソサミンを植物により高度に生産することが可能になる。 By using a transformed plant cell or a transformed plant that produces GFAT as described above, hexosamine can be produced to a high degree by the plant.
上記形質転換植物又は形質転換植物細胞を培養し、植物内にヘキソサミンを生産させた後、該形質転換植物細胞又は植物から適宜公知の方法によって、ヘキソサミンを抽出することができる。 After culturing the transformed plant or transformed plant cell and producing hexosamine in the plant, hexosamine can be extracted from the transformed plant cell or plant by a known method as appropriate.
形質転換植物又は形質転換植物細胞からの抽出において、植物体全体から抽出操作を行ってもよい。また、植物体を根、茎、葉と分別して抽出操作を行ってもよいし、それぞれを組み合わせて抽出操作を実施してもよい。 In extraction from a transformed plant or transformed plant cell, an extraction operation may be performed from the whole plant. Further, the plant body may be separated from roots, stems, and leaves, and the extraction operation may be performed, or each may be combined to perform the extraction operation.
形質転換植物又は形質転換植物細胞は、生の状態、凍結された状態、凍結乾燥された状態のいずれから抽出操作を行ってもよい。好ましくは、生の状態から抽出作業を行うのがよい。生または凍結された形質転換植物又は形質転換植物細胞から抽出作業を行う場合には、まず、破砕をしても良い。破砕にはミキサー、ホモジナイザー、乳鉢等を用いて破砕すればよい。凍結乾燥されたものは、凍結乾燥操作後、粉状に破砕しても良い。
破砕された形質転換植物又は形質転換植物細胞は溶媒を用いて、抽出操作を行う。抽出方法として、室温，冷却又は加熱した状態で含浸させて抽出する方法、水蒸気蒸留等の蒸留法を用いて抽出する方法、圧搾して抽出物を得る圧搾法、超臨界流体又は亜臨界流体を用いて抽出する方法等を用いてもよく、これらの方法を単独で、又は２種以上を組み合わせて抽出を行っても良い。好ましくは、細かく裁断した原料１ｇに対して、５〜１００ｍｌの溶媒を用い、１時間から１か月間、好ましくは１〜５日間、室温或いは加熱下で行うことが望ましい。
抽出に用いられる溶媒は、ヘキソサミンが効果的に抽出される溶媒であれば特に限定されるものではないが、水または、エタノール等の低級アルコールのように水と混和する有機溶媒、またはそれらの混液、または水と混和しない有機溶媒たとえば酢酸エチル等を用いてもよい。そのほかにも例を挙げるならば、メタノール，イソプロパノール，イソブタノール，n-ヘキサノール，メチルアミルアルコール等の１価アルコール類、エチレングリコール，プロピレングリコール等の多価アルコール又はその誘導体、アセトン，メチルエチルケトン，等のケトン類，酢酸イソプロピル等のエステル類、エチルエーテル，イソプロピルエーテル等のエーテル類等が例示される。また、リン酸緩衝生理食塩水等の無機塩類を添加した極性溶媒、界面活性剤を添加した溶媒を用いることもでき、特に限定されない。
The transformed plant or transformed plant cell may be subjected to an extraction operation from any of a raw state, a frozen state, and a lyophilized state. Preferably, the extraction operation is performed from the raw state. When the extraction operation is performed from a live or frozen transformed plant or transformed plant cell, it may be first crushed. What is necessary is just to crush using a mixer, a homogenizer, a mortar, etc. for crushing. The freeze-dried product may be crushed into a powder after the freeze-drying operation.
The disrupted transformed plant or transformed plant cell is extracted using a solvent. As an extraction method, a method of extracting by impregnating at room temperature, in a cooled or heated state, a method of extracting using a distillation method such as steam distillation, a pressing method of obtaining an extract by pressing, a supercritical fluid or a subcritical fluid Extraction methods may be used, and these methods may be used alone or in combination of two or more. Preferably, 5 to 100 ml of solvent is used per 1 g of finely cut raw material, and it is desirable to carry out at room temperature or under heating for 1 hour to 1 month, preferably 1 to 5 days.
The solvent used for the extraction is not particularly limited as long as hexosamine can be effectively extracted, but water, an organic solvent miscible with water such as a lower alcohol such as ethanol, or a mixture thereof. Alternatively, an organic solvent immiscible with water, such as ethyl acetate, may be used. Other examples include monohydric alcohols such as methanol, isopropanol, isobutanol, n-hexanol and methyl amyl alcohol, polyhydric alcohols such as ethylene glycol and propylene glycol or derivatives thereof, acetone, methyl ethyl ketone, and the like. Examples include ketones, esters such as isopropyl acetate, and ethers such as ethyl ether and isopropyl ether. Moreover, the polar solvent which added inorganic salts, such as phosphate buffered saline, and the solvent which added surfactant can also be used, It does not specifically limit.
上記のヘキソサミンを含む抽出液を濾過後、植物細胞を含まないヘキソサミンの濾過溶液を得る。ヘキソサミンを含む濾過溶液を定法（イオン交換カラム、活性炭カラム、塩析等）により精製することができる。 After filtering the extract containing hexosamine, a filtered solution of hexosamine that does not contain plant cells is obtained. A filtered solution containing hexosamine can be purified by a conventional method (ion exchange column, activated carbon column, salting out, etc.).
また、本発明においては、形質転換植物細胞を培養し、その培地からから適宜公知の方法によって、ヘキソサミンを回収することができる。 In the present invention, transformed plant cells are cultured, and hexosamine can be recovered from the medium by appropriately known methods.
本発明におけるヘキソサミンはヘキソースにアミノ基が導入された化合物であれば限定されないが、グルコサミン、ガラクトサミン、マンノサミン、またはそれらのN-アセチル体であるＮ−アセチルグルコサミン(GlcNAc)、Ｎ−アセチルガラクトサミン(GalNAc)、Ｎ−アセチルマンノサミン(ManNAc)、またはそれらにリン酸基が付加されたグルコサミン−１−リン酸（GlcN-1-P）、グルコサミン−６−リン酸(GlcN-6-P)、Ｎ−アセチルグルコサミン−１−リン酸(GlcNAc-1-P)、Ｎ−アセチルグルコサミン−６−リン酸(GlcNAc-6-P)などが例示されるが、本発明はこれに限定されるものではない。 The hexosamine in the present invention is not limited as long as it is a compound in which an amino group is introduced into hexose. ), N-acetylmannosamine (ManNAc), or glucosamine-1-phosphate (GlcN-1-P), glucosamine-6-phosphate (GlcN-6-P) having a phosphate group added thereto, Examples include N-acetylglucosamine-1-phosphate (GlcNAc-1-P) and N-acetylglucosamine-6-phosphate (GlcNAc-6-P), but the present invention is not limited thereto. Absent.
以下に実施例を示して本発明を具体的に説明するが、本発明は実施例に限定されるものではない。
（実施例１） クロレラウイルス由来GFAT遺伝子の単離
クロレラウイルス由来グルタミン：フルクトース−６−リン酸 アミドトランスフェラーゼ遺伝子（cvGFAT）をPCRにより単離するためにPCRプライマーを作製した。既に明らかになっているクロレラウイルスPBCV1系統の配列情報を参考とし、GFAT推定領域の外側100bpから増幅できるように、配列番号６のプライマーＰ４、及び配列番号７のＰ５を設計した。
PCR は、DNAポリメラーゼにKOD −plus- （東洋紡）を用い、94℃ ２分、（94℃ 15秒、60℃ 30秒、68℃ 1分）30サイクルの反応プログラムで行った。鋳型DNAにはクロレラウイルス弘前系統（CVHIＩ）のゲノムDNAを使用した。得られたPCR断片をpCRscript（Stratagene製）にクローニングした。挿入された断片の配列を、DNAシークエンサーにより解読し、CVHI1株由来の新規なGFAT遺伝子を得、これを「cvGFAT-HI遺伝子」と命名した。cvGFAT-HI遺伝子のアミノ酸配列は配列番号１、遺伝子配列は配列番号２に示す。
EXAMPLES The present invention will be specifically described below with reference to examples, but the present invention is not limited to the examples.
Example 1 Isolation of Chlorella Virus-Derived GFAT Gene PCR primers were prepared to isolate the chlorella virus-derived glutamine: fructose-6-phosphate amide transferase gene (cvGFAT) by PCR. The primer P4 of SEQ ID NO: 6 and the P5 of SEQ ID NO: 7 were designed so that amplification was possible from the outer 100 bp of the GFAT predicted region with reference to the already known sequence information of the chlorella virus PBCV1 strain.
PCR was performed using a reaction program of 94 cycles at 94 ° C. for 2 minutes and 94 ° C. for 15 seconds, 60 ° C. for 30 seconds, 68 ° C. for 1 minute using KOD-plus- (Toyobo) as the DNA polymerase. As template DNA, genomic DNA of the chlorella virus Hirosaki strain (CVHII) was used. The obtained PCR fragment was cloned into pCRscript (Stratagene). The sequence of the inserted fragment was decoded with a DNA sequencer to obtain a novel GFAT gene derived from the CVHI1 strain, which was named “cvGFAT-HI gene”. The amino acid sequence of the cvGFAT-HI gene is shown in SEQ ID NO: 1, and the gene sequence is shown in SEQ ID NO: 2.
（実施例２） 無細胞発現ベクターへのクローニング
配列番号2で示されるcvGFAT-HI遺伝子のオープンリーディングフレーム領域を小麦胚芽翻訳系（wheat germ extract）を用いた無細胞タンパク質合成用ベクターに導入し、タンパク質の発現を行った。具体的には、発現プラスミドの作製とタンパク質の発現には、「PROTEIOS Wheat germ cell-free protein synthesis core kit」（東洋紡製）を用い、添付された取扱説明書に従って行った。
(Example 2) Cloning into a cell-free expression vector The open reading frame region of the cvGFAT-HI gene represented by SEQ ID NO: 2 was introduced into a cell-free protein synthesis vector using a wheat germ translation system, Protein expression was performed. Specifically, “PROTEIOS Wheat germ cell-free protein synthesis core kit” (manufactured by Toyobo Co., Ltd.) was used for the production of expression plasmids and protein expression, according to the attached instruction manual.
cvGFAT-HI遺伝子を含むpCRscriptを鋳型に配列番号３で示されるプライマーＰ１と配列番号４で示されるプライマーＰ２を用い、前記条件でPCRを行った。PCR は、DNAポリメラーゼにKOD −plus-を用い、94℃ ２分、（94℃ 15秒、60℃ 30秒、68℃ 1分）30サイクルの反応プログラムで行った。PCR産物をＸｂａＩ切断処理し、pEU-NIIベクターのＥｃｏＲＶサイトとＸｂａＩサイトにクローニングした。該連結反応液を用いてEscherichia coli DH5α株を前述の公知の方法に従って形質転換し、該形質転換体をアンピシリン（50μg/ml）を含むLuria-Bertani Medium（ＬＢ）寒天培地（バクトトリプトン 10 g/l、酵母エキス 10 g/l、塩化ナトリウム 5g/l、寒天 15g/l）に塗布後、３７℃で一晩培養した。生育してきた形質転換体のコロニーより公知の方法に従ってプラスミドを抽出した。以上より、プラスミドpEU／cvGFAT-HIを構築した。 PCR was performed under the above conditions using primer P1 represented by SEQ ID NO: 3 and primer P2 represented by SEQ ID NO: 4 using pCRscript containing the cvGFAT-HI gene as a template. PCR was carried out using a reaction program of 94 ° C for 2 minutes and 94 ° C for 15 seconds, 60 ° C for 30 seconds, 68 ° C for 1 minute) using KOD-plus- as the DNA polymerase. The PCR product was digested with XbaI and cloned into the EcoRV site and the XbaI site of the pEU-NII vector. Escherichia coli DH5α strain was transformed with the ligation reaction solution according to the above-mentioned known method, and the transformant was transformed into Luria-Bertani Medium (LB) agar medium (bactotryptone 10 g containing ampicillin (50 μg / ml). / l, yeast extract 10 g / l, sodium chloride 5 g / l, agar 15 g / l) and cultured at 37 ° C. overnight. A plasmid was extracted from the grown colonies of the transformant according to a known method. From the above, plasmid pEU / cvGFAT-HI was constructed.
（実施例３） GFATタンパク質の発現
pEU／cvGFAT-HIを用いてmRNAの合成を行い、cvGFAT-HI遺伝子のオープンリーディングフレームを発現させた。具体的には、5μgのプラスミドpEU／cvGFAT-HIを鋳型としてT7 RNA Polymeraseで37℃、４時間反応を行い、mRNAを合成した。その後、6μgのmRNAをwheat germ extractと混合し、重曹法で26℃、24時間反応した。反応溶液をサンプルバッファー（50mM Tris-HCl (pH6.8)、2% SDS、10% グリセロール、0.6% βメルカプトエタノール）に懸濁し、5分間煮沸した後、発現したタンパク質をSDS-PAGEにて確認した。
(Example 3) Expression of GFAT protein
mRNA was synthesized using pEU / cvGFAT-HI to express the open reading frame of the cvGFAT-HI gene. Specifically, 5 μg of plasmid pEU / cvGFAT-HI was used as a template and reacted with T7 RNA Polymerase at 37 ° C. for 4 hours to synthesize mRNA. Thereafter, 6 μg of mRNA was mixed with wheat germ extract and reacted at 26 ° C. for 24 hours by the sodium bicarbonate method. The reaction solution was suspended in sample buffer (50 mM Tris-HCl (pH 6.8), 2% SDS, 10% glycerol, 0.6% β mercaptoethanol), boiled for 5 minutes, and the expressed protein was confirmed by SDS-PAGE. did.
（実施例４） GFAT活性の確認
タンパク質発現液をGFAT反応に供試した。具体的にはフルクトース−６−リン酸（15mM）、L-グルタミン（15mM）、EDTA(1mM)、DTT(1mM)、KH2PO4(60mM、pH7.0)を含む溶液にタンパク質発現液50μlを添加して、37℃で4時間反応させた。Morgan&Elson法の改良法であるReissig法（J.Biol.Chem、1955）を用いて測定し、A544を測定した。結果を図１に示す。コントロールであるpEU／DHFRではA544は検出されなかったが、pEU／cvGFAT-HIではA544の増加が見られ、タンパク質発現液の中に活性型のGFAT酵素が存在することが確認された。
(Example 4) Confirmation of GFAT activity A protein expression solution was subjected to a GFAT reaction. Specifically, 50 μl of protein expression solution is added to a solution containing fructose-6-phosphate (15 mM), L-glutamine (15 mM), EDTA (1 mM), DTT (1 mM), KH2PO4 (60 mM, pH 7.0). And reacted at 37 ° C. for 4 hours. A 544 was measured using the Reissig method (J. Biol. Chem, 1955), which is an improved method of the Morgan & Elson method. The results are shown in FIG. In the control pEU / DHFR, A 544 was not detected, but in pEU / cvGFAT-HI, an increase in A 544 was observed, confirming the presence of active GFAT enzyme in the protein expression solution.
（実施例５） cvGFAT植物発現用ベクターの作製
全長のＧＦＡＴ遺伝子を増幅する配列番号５で示されるプライマ−Ｐ３を設計した。発現ベクターへの導入に必要な制限酵素部位として、5'側プライマーにBamHI部位を付加した。
(Example 5) Production of cvGFAT plant expression vector Primer P3 shown in SEQ ID NO: 5 was designed to amplify the full-length GFAT gene. A BamHI site was added to the 5 ′ primer as a restriction enzyme site necessary for introduction into the expression vector.
cvGFAT-HIを含むプラスミドDNAを鋳型として、プライマ−Ｐ２、Ｐ３を用い、上記プラスミドを鋳型としてPCRを行った。KOD −plus-を用い、94℃ ２分、（94℃ 15秒、50℃ 30秒、68℃ 1分）２サイクル、（94℃ 15秒、60℃ 30秒、68℃ 1分）28サイクルの反応プログラムで行った。PCRで得られた断片をBamHＩで処理した。 PCR was performed using the plasmid DNA containing cvGFAT-HI as a template, primers P2 and P3, and the plasmid as a template. Using KOD-plus-, 94 ° C for 2 minutes, 94 ° C for 15 seconds, 50 ° C for 30 seconds, 68 ° C for 1 minute), 2 cycles (94 ° C for 15 seconds, 60 ° C for 30 seconds, 68 ° C for 1 minute) for 28 cycles The reaction program was performed. The fragment obtained by PCR was treated with BamHI.
続いて、発現用組換えベクターpBI121(Jefferson et al., 1987, EMBO J, 6, 3901-3907)へcvGFAT-HIを挿入するため、pBI121を制限酵素SacIで処理後、Blunting High(東洋紡製)で平滑処理した後、BamHＩで処理し、上記の制限酵素処理したcvGFAT-HI遺伝子をクローニングした。以上より、cvGFAT-HIを含むpBI121プラスミド（pBI121 /cvGFAT-HI）が作製できた。 Subsequently, in order to insert cvGFAT-HI into the recombinant vector for expression pBI121 (Jefferson et al., 1987, EMBO J, 6, 3901-3907), pBI121 was treated with the restriction enzyme SacI and then Blunting High (manufactured by Toyobo) The cvGFAT-HI gene treated with BamHI and treated with the restriction enzyme was cloned. From the above, a pBI121 plasmid (pBI121 / cvGFAT-HI) containing cvGFAT-HI was prepared.
（実施例６） エレクトロポレーションコンピテントセルの作製
アグロバクテリウムLBA4404（Agrobacterium tumefaciens strain LBA4404）の単一コロニーを5 mLのLB培地に植菌し、28℃で1晩振盪培養した。この培養液を、500mLのLB培地に植菌し、600 nmにおける濁度が 0.5になるまで 28 ℃で振盪培養した。培養液を遠心分離 (5000 rpm, 10 min, 4 ℃)により集菌して上清を除去し、菌体を洗浄するため 500 mLの滅菌水を加えて懸濁し、再度遠心分離 (5000 rpm, 10 min, 4 ℃) により集菌して上清を除去した。この操作を 2回繰り返した後、沈殿に 20 mLの冷却した滅菌 10%グリセロール溶液を加えて懸濁し、遠心分離 (5000 rpm, 10 min, 4 ℃) により集菌して上清を除去した。沈殿に3 mLの冷却した滅菌 10%グリセロール溶液を加えて懸濁し、40μLずつ1.5mL遠心管に分注して、液体窒素で凍結させてから -80℃で保存した。
(Example 6) Preparation of electroporation competent cell A single colony of Agrobacterium LBA4404 (Agrobacterium tumefaciens strain LBA4404) was inoculated into 5 mL of LB medium, and cultured with shaking at 28 ° C overnight. This culture solution was inoculated into 500 mL of LB medium and cultured at 28 ° C. with shaking until the turbidity at 600 nm reached 0.5. The culture is collected by centrifugation (5000 rpm, 10 min, 4 ° C), the supernatant is removed, 500 mL of sterile water is added to suspend the cells, and the suspension is centrifuged again (5000 rpm, The cells were collected at 10 min, 4 ° C) and the supernatant was removed. After this operation was repeated twice, 20 mL of a cooled sterilized 10% glycerol solution was added to the precipitate to suspend it, and the supernatant was removed by collecting the cells by centrifugation (5000 rpm, 10 min, 4 ° C.). The precipitate was suspended by adding 3 mL of a chilled sterilized 10% glycerol solution, and 40 μL each was dispensed into a 1.5 mL centrifuge tube, frozen in liquid nitrogen, and stored at −80 ° C.
（実施例７） アグロバクテリウムLBA4404株へのpBI121 /cvGFAT-HIの導入
A.tumefaciens LBA4404のエレクトロポレーションコンピテントセル40μlに発現プラスミドpBI121 /cvGFAT-HI（200μg/ml）1μlを混合した懸濁液を、あらかじめ氷中で冷却した電極間距離1mmのキュベットに注入し、パルス電場（1.8kV、25μF、200Ω）を印加した。直ちにSOC500μlを加え、28℃にて３時間培養した後、カナマイシンを含むLB寒天培地に塗布し、25℃で３日間培養し、pBI121 /cvGFAT-HIを含むアグロバクテリウムを得た。
(Example 7) Introduction of pBI121 / cvGFAT-HI into Agrobacterium strain LBA4404
A suspension of 1 μl of expression plasmid pBI121 / cvGFAT-HI (200 μg / ml) mixed with 40 μl of electroporation competent cell of A. tumefaciens LBA4404 was injected into a cuvette with a distance of 1 mm between electrodes previously cooled in ice, A pulsed electric field (1.8 kV, 25 μF, 200Ω) was applied. Immediately after adding 500 μl of SOC and culturing at 28 ° C. for 3 hours, it was applied to an LB agar medium containing kanamycin and cultured at 25 ° C. for 3 days to obtain Agrobacterium containing pBI121 / cvGFAT-HI.
（実施例８） pBI121 /cvGFAT-HIを含むアグロバクテリウムLBA4404株によるタバコ培養細胞（BY-2）の感染
形質転換タバコ培養細胞は、Nicotiana tabacum L. cv Bright Yellow 2 (以下、BY-2と表すことがある。Nagata et al., 1981, Mol. Gen. Genet., 184, 161-165) を使用し,タバコ培養細胞の培養は、Nagataらの方法 （Nagata et al., 1981, Mol. Gen. Genet., 184,161-165）に従い、LS（Linsmaier and Skoog）培地（Linsmaier and Skoog, 1965, Physiol. Plant., 18, 100-127）中のKH2PO4を370mg/L、thiamine HClを1mg/Lに増量し、さらに最終濃度3%の sucroseおよび最終濃度0.2 mg/Lの ２，４−ジクロロフェノキシ酢酸（2,4-D）を添加した改変LS培地で行った。
(Example 8) Infection of tobacco cultured cells (BY-2) with Agrobacterium LBA4404 strain containing pBI121 / cvGFAT-HI Transformed tobacco cultured cells were obtained from Nicotiana tabacum L. cv Bright Yellow 2 (hereinafter referred to as BY-2). Using Nagata et al., 1981, Mol. Gen. Genet., 184, 161-165), tobacco cultured cells were cultured by the method of Nagata et al. (Nagata et al., 1981, Mol. Gen. Genet., 184,161-165), 370 mg / L of KH2PO4 and 1 mg / L of thiamine HCl in LS (Linsmaier and Skoog, 1965, Physiol. Plant., 18, 100-127) The test was carried out in a modified LS medium supplemented with sucrose having a final concentration of 3% and 2,4-dichlorophenoxyacetic acid (2,4-D) having a final concentration of 0.2 mg / L.
タバコ培養細胞の形質転換は、基本的にAnの方法（An, 1985, Plant Physiol., 79, 568-570）に従った。カナマイシン50mg/Lを含む5mLのLB 培地で28℃にて1晩培養したpBI121/cvGFAT-HIを含むそれぞれのアグロバテリウム培養液100μLと、培養4日目のタバコ培養細胞懸濁液4mLをシャーレに入れてよく混ぜ、25℃で２晩、暗所下で静置して共存培養した
。アグロバクテリウムを除くため、シャーレの中の培養液を50mLの遠心管に移して、さらに、カナマイシン（100mg/L）とカルベニシリン（250mg/L）を含む改変LS培地を20mL加えて、遠心（1000rpm,４分）し、上清を除去した。直ちに、新しい改変LS培地25mLを入れて遠心（1000rpm,４分）し、細胞を洗浄した。この操作を3回繰り返し、アグロバクテリウムを除いた培養細胞をカナマイシン（100mg/L）とカルベニシリン（250mg/L）を含む改変LS固形培地上に塗布し、25℃で暗黒下に静置して培養した。約2-3週間後にカルス化した細胞を新しい改変LS固形培地上に移植し、増殖しているクローンを選択した。選択した複数のクローンをカナマイシン（100mg/L）とカルベニシリン（250mg/L）を含む改変LS培地30mLに移し、継代培養を行った。
The transformation of tobacco cultured cells basically followed the method of An (An, 1985, Plant Physiol., 79, 568-570). Transfer 100 μL of each agrobaterium culture medium containing pBI121 / cvGFAT-HI cultured overnight at 28 ° C in 5 mL of LB medium containing 50 mg / L of kanamycin and 4 mL of tobacco culture cell suspension on the 4th day of culture. And mixed well, and allowed to stand at 25 ° C for 2 nights in the dark and cocultured. To remove Agrobacterium, transfer the culture medium in the petri dish to a 50 mL centrifuge tube, add 20 mL of modified LS medium containing kanamycin (100 mg / L) and carbenicillin (250 mg / L), and centrifuge (1000 rpm). 4 minutes) and the supernatant was removed. Immediately, 25 mL of new modified LS medium was added and centrifuged (1000 rpm, 4 minutes) to wash the cells. Repeat this procedure three times, and apply the cultured cells excluding Agrobacterium on a modified LS solid medium containing kanamycin (100 mg / L) and carbenicillin (250 mg / L), and leave it at 25 ° C in the dark. Cultured. About 2-3 weeks later, the callus cells were transplanted onto a new modified LS solid medium, and proliferating clones were selected. The selected clones were transferred to 30 mL of a modified LS medium containing kanamycin (100 mg / L) and carbenicillin (250 mg / L), and subcultured.
（実施例９） HPAEC（high-performance anion-exchange chromatography）法によるcvGFAT-HI組換えタバコ培養細胞（BY-2）の糖分析
液体培養中のタバコ培養細胞から、ヌッチェ濾過により培地を除去した。液体窒素で凍結した細胞を乳棒・乳鉢で破砕し、1/4量の滅菌水を加え、直ちに熱処理した。熱処理サンプルを遠心ろ過チューブ（孔径0.2μm）に通し、細胞内粗抽出液として、糖類分析システムDXc-500（日本ダイオネクス社製）を用いた解析に供試した。測定カラムにはCarboPac PA1（日本ダイオネクス社製、250mm x 4mm I.D.）を用い、移動相A（0.1M水酸化ナトリウム）および移動相B（0.1M水酸化ナトリウム、1M 酢酸ナトリウム）を0-40min（B.conc 5%→20%）、40min-60min（B.conc 20%）のグラジェントモードで、35℃にて流速1ml/minで流し、電気化学検出器ED-50A（日本ダイオネクス社製）により、溶出した糖を検出した。
図2は、結果の一例を示す。非形質転換培養細胞とcvGFAT-HI組換え培養細胞の細胞内粗抽出液を比較したところ、cvGFAT-HI組換え培養細胞において、GlcN-6-Pのピークが特異的に検出された。
(Example 9) Sugar analysis of cvGFAT-HI recombinant tobacco cultured cell (BY-2) by HPAEC (high-performance anion-exchange chromatography) method The medium was removed by Nutsche filtration from tobacco cultured cells in liquid culture. Cells frozen with liquid nitrogen were crushed with a pestle and mortar, and 1/4 amount of sterilized water was added and immediately heat-treated. The heat-treated sample was passed through a centrifugal filtration tube (pore size: 0.2 μm) and subjected to analysis using a saccharide analysis system DXc-500 (manufactured by Nippon Dionex) as a crude intracellular extract. CarboPac PA1 (Nihon Dionex, 250 mm x 4 mm ID) was used for the measurement column, and mobile phase A (0.1 M sodium hydroxide) and mobile phase B (0.1 M sodium hydroxide, 1 M sodium acetate) were added for 0-40 min ( B.conc 5% → 20%), 40min-60min (B.conc 20%) gradient mode, flow at 35 ° C at a flow rate of 1ml / min, electrochemical detector ED-50A (manufactured by Nippon Dionex) Was used to detect the eluted sugar.
FIG. 2 shows an example of the results. When the intracellular crude extracts of non-transformed cultured cells and cvGFAT-HI recombinant cultured cells were compared, the peak of GlcN-6-P was specifically detected in the cvGFAT-HI recombinant cultured cells.
（実施例１０） ABEE標識法によるcvGFAT-HI組換えタバコ培養細胞（BY-2）の糖分析
液体培養中のタバコ培養細胞から、ヌッチェ濾過により培地を除去した。培地を除去した細胞に1/4量の滅菌水、ガラスビーズ（直径1mm）100μlを加え、BeadSmash（和研薬BS-12）を用いて3,000rpm,3minで細胞を破砕した。遠心分離により上清を回収し、100℃、5分の熱処
理をした。さらに、遠心分離により上清を細胞内粗抽出液として回収した。細胞内粗抽出液の糖を、ABEE（4-アミノ安息香酸エチルエステル）糖組成分析キット（Jオイルミルズ社製）を用いて、ABEEで蛍光標識した後、滅菌水にて10倍希釈し、20μlを高速液体クロマトグラフィー（ＨＰＬＣ）の測定サンプルとした。ＨＰＬＣの測定にはホーネンパックＣ１８（Jオイルミルズ社製、75mm x 4.6mm I.D.）を用い、移動相A（0.2Mホウ酸カリウム緩衝液（pH8.9）/アセトニトリル(93:7)）で30分間分析した後、移動相B（0.02％トリフルオロ酢酸/アセトニトリル(50:50)）で5分間洗浄し、さらに移動相Aで15分間平衡化した。ABEE標識糖は、30℃にて、移動相Aを流速1ml/minで流し、励起波長305nm、検出波長360nmの蛍光をモニターして分析した。図3は、細胞内粗抽出液のABEE標識糖を分析したＨＰＬＣの結果の一例を示す。非形質転換培養細胞とcvGFAT-HI組換え培養細胞の細胞内粗抽出液を比較した結果、cvGFAT-HI組換え培養細胞に特異的なピークが認められた。ABEE標識した糖をコントロールとして比較したところ、これらのピークがGlcNAc、GlcNAc-6-Pであることが確認された。細胞内粗抽出液中のGlcNAc濃度は0.2mMであった。
(Example 10) Sugar analysis of cvGFAT-HI recombinant tobacco cultured cells (BY-2) by ABEE labeling method The culture medium was removed from Nutsche filtration from tobacco cultured cells in liquid culture. 1/4 volume of sterilized water and 100 μl of glass beads (
また、液体培養中の培地画分を回収した後、100℃、5分の熱処理をし、遠心分離により上清を回収した。上記同様に、上清の糖をABEE標識し、HPLCにより測定したところ、培地中のGlcNAc濃度は0.3mMであることが示された。 In addition, after collecting the medium fraction during liquid culture, heat treatment was performed at 100 ° C. for 5 minutes, and the supernatant was collected by centrifugation. As described above, the supernatant sugar was labeled with ABEE and measured by HPLC, which showed that the GlcNAc concentration in the medium was 0.3 mM.
（実施例１１） pBI121/cvGFAT-HIを含むアグロバクテリウムLBA4404株によるタバコの感染
pBI121/cvGFAT-HIを含むアグロバクテリウムLBA4404株によるタバコ(Nicotiana tabacum SR-1)の形質転換は、アグロバクテリウムを用いるリーフディスク法（山田康之、岡田吉美編、「植物バイオテクノロジーII」、東京化学同人、1991年）に従った。カナマイシン50mg/Lを含む5mLのLB 培地で28℃にて1晩培養したpBI121cvHIを含むアグロバテリウムの培養液に、滅菌処理したタバコのリーフディスクを３分間浸した後、濾紙上で余分な菌体を除去し、 MS（Murashige and Skoog）無機塩（Murashige and Skoog, 1962, Physiol. Plant., 15, 473）に3% スクロース、B5ビタミン、1mg/L ベンジルアミノプリン、1mg/L ナフタレン酢酸および0.3% ゲランガムを添加し、pH5.7に調整した 分化培地に静置し、28℃にて２日間、暗所で静置した。感染させたリーフディスクを滅菌水で３回洗浄し、濾紙上で余分な水分を除去した後、抗生物質としてカナマイシン(100mg/L)およびクラフォラン(250mg/L)を含む分化培地に静置し、25℃にて16時間光条件下でカルスの形成を誘導した。誘導開始３週間後に、形態的に正常なシュートを選び、茎葉を含んだ状態で切り出し、カナマイシン(100mg/L)およびクラフォラン(250mg/L)を含む発根培地(MS無機塩、3% スクロース、B5ビタミンおよび0.3% ゲランガム、pH5.7)に移し、25℃にて16時間光条件下で発根を誘導した。２週間後に発根が認められたシュートを新鮮な発根培地に移し替え、茎葉の生育した複数個のラインが得られた。
(Example 11) Infection of tobacco by Agrobacterium LBA4404 strain containing pBI121 / cvGFAT-HI
Tobacco (Nicotiana tabacum SR-1) transformation by Agrobacterium LBA4404 strain containing pBI121 / cvGFAT-HI is based on leaf disk method using Agrobacterium (Yasuyuki Yamada, Yoshimi Okada, “Plant Biotechnology II”, Tokyo) Chemistry Doujin, 1991). After immersing sterilized tobacco leaf discs in agrobaterium containing pBI121cvHI grown in 5 mL LB medium containing kanamycin 50 mg / L at 28 ° C overnight, excess bacteria on filter paper The body is removed and MS (Murashige and Skoog) inorganic salt (Murashige and Skoog, 1962, Physiol. Plant., 15, 473) is added with 3% sucrose, B5 vitamin, 1 mg / L benzylaminopurine, 1 mg / L naphthalene acetic acid and 0.3% gellan gum was added and the mixture was allowed to stand in a differentiation medium adjusted to pH 5.7, and allowed to stand at 28 ° C. for 2 days in the dark. The infected leaf disk was washed with sterilized water three times, and after removing excess water on the filter paper, it was allowed to stand in a differentiation medium containing kanamycin (100 mg / L) and claforan (250 mg / L) as antibiotics. Callus formation was induced under light conditions at 25 ° C for 16 hours. Three weeks after the start of induction, morphologically normal shoots were selected, cut out with foliage, and rooting medium (MS inorganic salt, 3% sucrose, containing kanamycin (100 mg / L) and claforan (250 mg / L), B5 vitamin and 0.3% gellan gum, pH 5.7) and induced rooting under light conditions at 25 ° C. for 16 hours. After two weeks, shoots with rooting were transferred to a fresh rooting medium, and a plurality of lines with foliage were obtained.
（実施例１２） cvGFAT-HI組換えタバコの糖分析
上述のアグロバクテリウムによる感染で得られた10ラインの形質転換タバコの葉約100mgを2ml容のチューブに移し、100μlのバッファー (20mM Tris-HCl pH7.5)を加えて懸濁し、400mgのステンレスビーズ(直径4.8mm)を加えた。ビードスマッシュ(和研薬 BS-12)を用いて、チューブを振とう攪拌処理することにより、タバコの葉を破砕処理した(4,000rpm, 1分)。遠心分離により上清を回収し、100℃、5分の熱処理をした。さらに、遠心分離により上清を細胞内粗抽出液として回収した。引き続き、実施例10と同様に、ABEE糖組成分析キットを用いて、ＨＰＬＣにより細胞内粗抽出液のヘキソサミンを分析した。その結果、細胞内粗抽出液にGlcNAcが検出された。尚、非形質転換体のタバコの葉を用いて同様に分析したが、GlcNAcは検出されず、該形質転換体においてGlcNAcが高度に蓄積されていることが示された。
Example 12 Sugar Analysis of cvGFAT-HI Recombinant Tobacco Approximately 100 mg of 10 lines of transformed tobacco obtained by infection with Agrobacterium described above was transferred to a 2 ml tube, and 100 μl of buffer (20 mM Tris- HCl pH 7.5) was added and suspended, and 400 mg of stainless beads (diameter 4.8 mm) were added. Using a bead smash (Waken Pharmaceutical BS-12), the tube was shaken and stirred to crush tobacco leaves (4,000 rpm, 1 minute). The supernatant was collected by centrifugation and heat-treated at 100 ° C. for 5 minutes. Furthermore, the supernatant was recovered as an intracellular crude extract by centrifugation. Subsequently, as in Example 10, hexosamine in the intracellular crude extract was analyzed by HPLC using an ABEE sugar composition analysis kit. As a result, GlcNAc was detected in the intracellular crude extract. The same analysis was performed using non-transformed tobacco leaves, but GlcNAc was not detected, indicating that GlcNAc was highly accumulated in the transformant.
（実施例１３） 形質転換タバコ（T1世代）の糖分析
実施例１１で作製したcvGFAT遺伝子を導入した形質転換タバコから種子を採取し、カナマイシン(100mg/L)を含むMS分化培地に播種した。成長した形質転換タバコ（T1世代）から実施例１２に示した方法と同様に粗抽出液を調製し、ABEE糖組成分析キットを用いて、ＨＰＬＣにより細胞内粗抽出液のヘキソサミンを分析した。その結果、T0世代同様にGlcNAcが高度に蓄積していることが確認された。
(Example 13) Sugar analysis of transformed tobacco (T1 generation) Seeds were collected from the transformed tobacco introduced with the cvGFAT gene prepared in Example 11 and sown in an MS differentiation medium containing kanamycin (100 mg / L). A crude extract was prepared from the grown transformed tobacco (T1 generation) in the same manner as in Example 12, and hexosamine in the intracellular crude extract was analyzed by HPLC using an ABEE sugar composition analysis kit. As a result, it was confirmed that GlcNAc was highly accumulated as in the T0 generation.
本発明によると、植物細胞または植物体から有用なGlcNAc、GlcNAc-6-Pなどのヘキソサミンを安全、安価に、低環境負荷で生産することができる。特に、GlcNAcは、甘味料としての用途だけではなく、変形関節炎の治療を目的とした医薬用途にも利用されており、本発明により食品分野、医薬分野の研究開発を活性化でき、産業界に大きく寄与することができる。 According to the present invention, hexosamine such as GlcNAc and GlcNAc-6-P useful from plant cells or plants can be produced safely and inexpensively with a low environmental load. In particular, GlcNAc is used not only as a sweetener, but also for pharmaceutical use for the treatment of osteoarthritis. The present invention can activate research and development in the food and pharmaceutical fields, and can be used in industry. It can contribute greatly.
Claims (22)
（２）形質転換して得られた形質転換体（形質転換植物細胞又は形質転換植物）を生育する工程、
（３）該形質転換体により生産されたヘキソサミンを分離する工程、
を含むヘキソサミンの製造方法。 (1) a step of transforming a plant cell with a recombinant expression vector, wherein an exogenous GFAT (glutamine: fructose-6-phosphate amide transferase) gene is introduced;
(2) a step of growing a transformant (transformed plant cell or transformed plant) obtained by transformation,
(3) a step of separating hexosamine produced by the transformant,
A method for producing hexosamine, comprising:
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