JP5637343B2 - Genetically modified tomato plant cultivation equipment - Google Patents

Genetically modified tomato plant cultivation equipment Download PDF

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JP5637343B2
JP5637343B2 JP2008291219A JP2008291219A JP5637343B2 JP 5637343 B2 JP5637343 B2 JP 5637343B2 JP 2008291219 A JP2008291219 A JP 2008291219A JP 2008291219 A JP2008291219 A JP 2008291219A JP 5637343 B2 JP5637343 B2 JP 5637343B2
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genetically modified
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tomato plant
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nutrient solution
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JP2010115158A (en
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直也 福田
直也 福田
浩 江面
浩 江面
剛 溝口
剛 溝口
京子 棚瀬
京子 棚瀬
正良 平井
正良 平井
英男 角田
英男 角田
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University of Tsukuba NUC
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Description

本発明は、例えば、植物の大量生産に用いられる植物体栽培装置及び遺伝子組換えトマトにおける目的タンパク質の効率的な生産方法に関する。   The present invention relates to a plant body cultivation apparatus used for mass production of plants and a method for efficiently producing a target protein in a genetically modified tomato, for example.

従来においては、閉鎖空間内の人工光照射下でトマトを生育させ、果実を収穫する実用的技術が存在しなかった。このような技術が存在すれば、外部環境の影響を受けることなく計画的にトマトを生産することが可能である。   In the past, there was no practical technique for growing tomatoes and harvesting fruits under artificial light irradiation in a closed space. If such a technique exists, it is possible to produce tomatoes systematically without being affected by the external environment.

一方、従来において、遺伝子組換えトマトにおける目的タンパク質の生産及び蓄積を最適なものとする光環境条件は検討されていなかった。このような光環境条件を設定できれば、遺伝子組換えトマトにおいて目的タンパク質を効率的に生産することできる。   On the other hand, conventionally, the light environment condition that optimizes the production and accumulation of the target protein in the genetically modified tomato has not been studied. If such light environment conditions can be set, the target protein can be efficiently produced in the genetically modified tomato.

例えば、本願発明者は、ミラクリンを発現する遺伝子組換え植物の作製方法を既に見出している(特許文献1)。ミラクリンは、ミラクルフルーツ(Richadella dulcifica)果実中に含まれる食味修飾タンパク質である。ミラクリンには食品中の酸味を甘味に変える働きがあり、例えば、ミラクルフルーツを食べた後にレモン果実を食べると、オレンジ果実のような甘みを感じる。このようなミラクリンの機能を利用して、ミラクリンを、体重制限のためのダイエットや糖尿病治療のための食事制限を無理なく行い、食事におけるカロリー摂取を低減するための補助食品として利用することが考えられている。   For example, the present inventor has already found a method for producing a genetically modified plant that expresses miraculin (Patent Document 1). Miracrine is a taste-modifying protein contained in the fruit of Richaella dulcifica. Miracrine has the function of changing the acidity in food to sweetness. For example, if you eat lemon fruit after eating miracle fruit, you will feel sweetness like orange fruit. Using such a function of miraculin, miraculin can be used as a dietary supplement to reduce dietary calorie intake by dieting for weight restriction and dietary treatment for diabetes. It has been.

このように、ミラクリン等のタンパク質を遺伝子組換え植物で生産及び蓄積させることは望まれる。   Thus, it is desirable to produce and accumulate proteins such as miraculin in transgenic plants.

国際公開第2006/014018号パンフレットInternational Publication No. 2006/014018 Pamphlet

そこで、本発明は、上述した実情に鑑み、閉鎖空間型植物体栽培装置及び当該装置を利用した、遺伝子組換えトマトにおける目的タンパク質の効率的な生産方法を提供することを目的とする。   Then, in view of the situation mentioned above, this invention aims at providing the efficient production method of the target protein in a genetically modified tomato using the closed space type plant body cultivation apparatus and the said apparatus.

上記課題を解決するため鋭意研究を行った結果、閉鎖系環境における特定の光環境条件下で、遺伝子組換えトマト内において当該遺伝子によりコードされるタンパク質が安定的に蓄積することを見出し、本発明を完成するに至った。   As a result of diligent research to solve the above problems, it was found that the protein encoded by the gene is stably accumulated in the genetically modified tomato under specific light environment conditions in a closed system environment. It came to complete.

すなわち、本発明は、植物体に対する光環境を調節する光環境調節手段と、光環境調節手段からの熱を調節する排気手段と、水耕栽培手段と、室内大気中炭酸ガス濃度調節手段とを備える栽培室を積層構造で複数備えることを特徴とする、植物体栽培装置である。   That is, the present invention comprises a light environment adjusting means for adjusting a light environment for a plant body, an exhaust means for adjusting heat from the light environment adjusting means, a hydroponic cultivation means, and an indoor atmospheric carbon dioxide concentration control means. A plant body cultivating apparatus comprising a plurality of cultivating rooms with a laminated structure.

また、本発明に係る植物体栽培装置は、水耕栽培手段に対して養液を供給及び回収する養液循環手段をさらに備えたものであってよい。   Moreover, the plant body cultivating apparatus according to the present invention may further include a nutrient solution circulation means for supplying and recovering the nutrient solution to the hydroponics means.

さらに、本発明に係る植物体栽培装置においては、上記光環境調節手段を植物体に対して上面及び側面の双方に配置することが好ましい。   Furthermore, in the plant cultivation apparatus which concerns on this invention, it is preferable to arrange | position the said light environment adjustment means to both an upper surface and a side surface with respect to a plant body.

本発明に係る植物体栽培装置において栽培する植物体としては、トマト植物体が挙げられる。   Examples of the plant cultivated in the plant cultivating apparatus according to the present invention include tomato plants.

また、本発明は、遺伝子組換えトマト植物体を明期6〜20時間未満、光合成有効光量子束密度に基づく光強度300〜600μmol/m2/s及び炭酸ガス濃度500〜1000ppmの条件下で栽培することを含む、導入遺伝子によりコードされるタンパク質を生産する方法である。当該導入遺伝子例としては、ミラクリンをコードする遺伝子が挙げられる。さらに、本発明に係る方法では、上述の本発明に係る植物体栽培装置を用いて、遺伝子組換えトマト植物体を栽培することが好ましい。 The present invention also cultivates a transgenic tomato plant under the conditions of light period of 6 to less than 20 hours, light intensity of 300 to 600 μmol / m 2 / s based on photosynthetic effective photon flux density, and carbon dioxide concentration of 500 to 1000 ppm. To produce a protein encoded by the transgene. Examples of the transgene include a gene encoding miraculin. Furthermore, in the method according to the present invention, it is preferable to cultivate a genetically modified tomato plant using the plant cultivating apparatus according to the present invention described above.

本発明に係る植物体栽培装置によれば、閉鎖空間内の人工光照射下で、安定的にトマト等の植物を大量に生産することができる。また、本発明に係る方法によれば、遺伝子組換えトマトにおいて、導入遺伝子によりコードされる目的タンパク質の蓄積が安定化し、有用タンパク質を大量生産することができる。   According to the plant cultivation apparatus concerning the present invention, plants such as tomatoes can be stably produced in large quantities under artificial light irradiation in a closed space. Further, according to the method of the present invention, accumulation of a target protein encoded by a transgene is stabilized in a genetically modified tomato, and useful proteins can be mass-produced.

以下、本発明を詳細に説明する。
図1に示すように、本発明に係る植物体栽培装置1は、植物体2に対する光環境を調節する光環境調節手段3と、光環境調節手段からの熱を調節する排気手段4と、水耕栽培手段5と、室内大気中炭酸ガス濃度調節手段(図1中には示されていない)とを備える栽培室6を積層構造で複数備える。
Hereinafter, the present invention will be described in detail.
As shown in FIG. 1, a plant cultivation apparatus 1 according to the present invention includes a light environment adjusting unit 3 that adjusts a light environment for a plant 2, an exhaust unit 4 that adjusts heat from the light environment adjusting unit, and water. A plurality of cultivation rooms 6 having a cultivation structure 5 and indoor atmospheric carbon dioxide concentration adjusting means (not shown in FIG. 1) are provided in a laminated structure.

ここで、植物体2とは、植物体全体、植物体の苗、器官(例えば葉、花弁、茎、根、種子等)、植物組織(例えば表皮、師部、柔組織、木部、維管束等)又は植物培養細胞を意味する。好ましくは、植物体2は苗である。また、植物体2としては、水耕栽培により栽培することができる植物体であればいずれの植物体であってもよく、例えば、トマト、キュウリ、イチゴ、ピーマン、レタス、チンゲンサイ、ホウレンソウ等の植物体が挙げられる。好ましくは、植物体2はトマト植物体である。   Here, the plant body 2 refers to the whole plant body, plant seedlings, organs (eg leaves, petals, stems, roots, seeds, etc.), plant tissues (eg epidermis, phloem, soft tissue, xylem, vascular bundles) Etc.) or plant cultured cells. Preferably, the plant body 2 is a seedling. The plant 2 may be any plant as long as it can be cultivated by hydroponics. For example, plants such as tomatoes, cucumbers, strawberries, peppers, lettuce, chingensai, and spinach The body is mentioned. Preferably, the plant body 2 is a tomato plant body.

光環境調節手段3としては、例えば蛍光灯の光源等が挙げられる。光環境調節手段3は、植物体2に対して均一に光を照射するために、植物体2に対して上面及び側面の双方に配置されることが好ましい。例えば、図2に示すように、光環境調節手段3(蛍光灯)が栽培室6の天井面と植物体2の配置に平行して(すなわち、栽培室6の側面から側面へ)配置されている。   Examples of the light environment adjusting means 3 include a fluorescent light source. The light environment adjusting means 3 is preferably arranged on both the upper surface and the side surface of the plant body 2 in order to uniformly irradiate the plant body 2 with light. For example, as shown in FIG. 2, the light environment adjusting means 3 (fluorescent lamp) is arranged in parallel with the ceiling surface of the cultivation room 6 and the arrangement of the plant body 2 (that is, from the side surface of the cultivation room 6 to the side surface). Yes.

排気手段4としては、例えば換気扇(ファン)が挙げられる。例えば、図2に示すように、排気手段4は、栽培室6の背面に配置される。   Examples of the exhaust means 4 include a ventilation fan (fan). For example, as shown in FIG. 2, the exhaust means 4 is disposed on the back surface of the cultivation room 6.

水耕栽培手段5は、植物体2を水耕栽培するための手段を意味する。例えば、水耕栽培手段5としては、水耕栽培用ベッド等が挙げられる。また、水耕栽培方法としては、薄膜水耕(Nutrient Film Technique;以下、「NFT」と呼ぶ)が挙げられる。NFTは、緩やかな傾斜を有する平面上に、養液を薄く(少量ずつ)流下させる水耕栽培を意味する。NFTにより植物体2を栽培すべく、例えば図3に示すように、植物体栽培装置1は、養液を供給及び回収する養液循環手段を備えることができる。養液循環手段では、循環タンク7から養液が給液10として供給され、水耕栽培手段5を通過した養液は排液11として循環タンク7に回収する。なお、循環タンク7内の養液量に応じて、養液は、養液自動調合装置8により、養液ストック9と水道水13から養液が自動制御で調合される。調合された養液は養液タンク12より、循環タンク7に回収された養液と共に水耕栽培手段5へと供給される。   The hydroponics means 5 means means for hydroponically cultivating the plant body 2. For example, hydroponics means 5 includes a hydroponics bed. As a hydroponics method, thin film hydroponics (hereinafter referred to as “NFT”) can be mentioned. NFT means hydroponics that causes the nutrient solution to flow down thinly (a little at a time) on a flat slope. In order to cultivate the plant body 2 by NFT, for example, as shown in FIG. 3, the plant body cultivation apparatus 1 can include nutrient solution circulation means for supplying and collecting the nutrient solution. In the nutrient solution circulation means, the nutrient solution is supplied from the circulation tank 7 as the feed solution 10, and the nutrient solution that has passed through the hydroponic cultivation means 5 is collected in the circulation tank 7 as the drainage solution 11. Note that, depending on the amount of nutrient solution in the circulation tank 7, the nutrient solution is prepared from the nutrient solution stock 9 and the tap water 13 by the nutrient solution automatic preparation device 8 by automatic control. The prepared nutrient solution is supplied from the nutrient solution tank 12 to the hydroponic cultivation means 5 together with the nutrient solution collected in the circulation tank 7.

室内大気中炭酸ガス濃度調節手段とは、栽培室6における炭酸ガス(二酸化炭素)濃度調節手段を意味する。当該室内大気中炭酸ガス濃度調節手段として、例えば炭酸ガス濃度測定手段と炭酸ガス濃度調節条件を制御する手段とを備えたものとすることができる。   The indoor atmospheric carbon dioxide concentration adjusting means means carbon dioxide (carbon dioxide) concentration adjusting means in the cultivation room 6. As the indoor atmospheric carbon dioxide concentration adjusting means, for example, a carbon dioxide concentration measuring means and a means for controlling the carbon dioxide concentration adjusting condition can be provided.

さらに、栽培室6には、室内温度調節手段を備えていてもよい。室内温度調節手段として、例えば温度測定手段と温度調節条件を制御する手段とを備えたものとすることができる。   Furthermore, the cultivation room 6 may be provided with an indoor temperature adjusting means. As the room temperature adjusting means, for example, a temperature measuring means and a means for controlling temperature adjusting conditions can be provided.

植物体栽培装置1においては、栽培室6が積層構造により複数(例えば、2、3、4、5個又はそれ以上)配置される。このように、植物体栽培装置1は、積層多段階式栽培装置とすることで、狭い閉鎖空間において単位床面積あたりにして大量に植物体を栽培することができる。   In the plant cultivation apparatus 1, a plurality of cultivation rooms 6 (for example, 2, 3, 4, 5 or more) are arranged in a laminated structure. Thus, the plant cultivation apparatus 1 can cultivate a large number of plants per unit floor area in a narrow closed space by using a stacked multi-stage cultivation apparatus.

以上に説明するように、本発明に係る植物体栽培装置によれば、閉鎖空間内の人工光照射下で、安定的に且つ連続的に植物体を大量に生産することができる。例えば、本発明に係る植物体栽培装置によれば、23〜40 kg/m2/年の収量でトマトを生産することができる。 As described above, according to the plant cultivation apparatus according to the present invention, a large number of plants can be stably and continuously produced under artificial light irradiation in a closed space. For example, according to the plant cultivation apparatus according to the present invention, tomatoes can be produced with a yield of 23 to 40 kg / m 2 / year.

また、本発明に係る植物体栽培装置では、植物体の状態に合わせて調光することも可能である。そこで、本発明に係る植物体栽培装置において、好適な光環境条件下で遺伝子組換えトマト植物体を栽培し、当該遺伝子組換えトマト植物体内において導入遺伝子によりコードされる目的タンパク質を大量に生産することができる。   Moreover, in the plant cultivation apparatus which concerns on this invention, it is also possible to adjust light according to the state of a plant. Therefore, in the plant cultivation apparatus according to the present invention, a genetically modified tomato plant is cultivated under suitable light environment conditions, and a large amount of a target protein encoded by the transgene is produced in the genetically modified tomato plant. be able to.

ここで、遺伝子組換えトマト植物体とは、外来遺伝子として導入された遺伝子(導入遺伝子)を発現しているか又は発現する状態で保持しているトマト植物体を意味する。遺伝子組換えトマト植物体は、植物に対する一般的な形質転換方法を用いて導入遺伝子で形質転換することにより作製することができる。例えば、形質転換方法としては、アグロバクテリウム法、エレクトロポレーション法、パーティクルガン法が挙げられる。これらの植物形質転換法の詳細は、『島本功、岡田清孝 監修「新版 モデル植物の実験プロトコール 遺伝学的手法からゲノム解析まで」(2001)秀潤社』等の一般的な教科書の記載や、Hiei Y.et al.,「Efficient transformation of rice(Oryza sativa L.)mediated by Agrobacterium and sequence analysis of the boundaries of the T−DNA.」, Plant J.(1994)6, 271-282等の文献を参照することができる。   Here, the genetically modified tomato plant body means a tomato plant body expressing or holding a gene introduced as a foreign gene (transgene). A genetically modified tomato plant can be prepared by transforming with a transgene using a general transformation method for plants. For example, examples of the transformation method include an Agrobacterium method, an electroporation method, and a particle gun method. For details on these plant transformation methods, refer to general textbooks such as “Isao Shimamoto, Kiyotaka Okada“ Experimental Protocols from Model Plants to Genetic Analysis ”(2001) Shujunsha” Hiei Y. et al. , “Efficient transformation of rice (Oryza sativa L.) mediated by Agrobacterium and sequence analysis of the boundaries of the T-DNA.”, Plant J. (1994) 6, 271-282 etc. can be referred.

導入遺伝子としては、例えば、ミラクリンをコードする遺伝子(以下、「ミラクリン遺伝子」という)等が挙げられる(特許文献1)。   Examples of the transgene include a gene encoding miraculin (hereinafter referred to as “miracrine gene”) (Patent Document 1).

本発明に係るタンパク質の生産方法では、遺伝子組換えトマト植物体を、例えば上述の本発明に係る植物体栽培装置において、所定の室内炭酸ガス濃度における短日強光条件下で栽培することで、トマト植物体内(例えば、トマト果実内)において導入遺伝子によりコードされる目的タンパク質を安定的に蓄積させることができる。短日強光条件としては、例えば、明期6〜20時間未満(好ましくは8〜20時間未満、特に好ましくは16時間)、且つ光合成有効光量子束密度に基づく光強度300〜600μmol/m2/s(好ましくは400〜600μmol/m2/s、特に好ましくは500μmol/m2/s)の条件が挙げられる。また、炭酸ガス濃度としては、例えば500〜1000ppmが挙げられる。なお、気温は、トマト植物体が生育する温度であればよく、例えば明期において20〜25℃(好ましくは23〜25℃、特に好ましくは25℃)、暗期において15〜20℃(好ましくは18〜20℃、特に好ましくは20℃)が挙げられる。 In the protein production method according to the present invention, a genetically modified tomato plant is cultivated under short-day intense light conditions at a predetermined indoor carbon dioxide concentration, for example, in the above-described plant cultivation apparatus according to the present invention, The target protein encoded by the transgene can be stably accumulated in the tomato plant body (for example, in the tomato fruit). As the short-day intense light conditions, for example, the light period is 6 to less than 20 hours (preferably less than 8 to 20 hours, particularly preferably 16 hours), and the light intensity based on the photosynthetic effective photon flux density is 300 to 600 μmol / m 2 / s (preferably 400 to 600 μmol / m 2 / s, particularly preferably 500 μmol / m 2 / s). Moreover, as carbon dioxide gas concentration, 500-1000 ppm is mentioned, for example. The temperature may be any temperature at which the tomato plant grows, for example, 20 to 25 ° C. (preferably 23 to 25 ° C., particularly preferably 25 ° C.) in the light period, and 15 to 20 ° C. (preferably in the dark period) 18 to 20 ° C., particularly preferably 20 ° C.).

本発明に係るタンパク質の生産方法では、当業者に周知のタンパク質抽出精製法を用いて、大量の目的タンパク質を取得することができる。すなわち、遺伝子組換えトマト植物体から目的タンパク質を含む抽出物を抽出し、好ましくはさらに目的タンパク質を分離することにより、目的タンパク質を得ることができる。   In the protein production method according to the present invention, a large amount of target protein can be obtained using protein extraction and purification methods well known to those skilled in the art. That is, the target protein can be obtained by extracting an extract containing the target protein from the transgenic tomato plant body, and preferably further separating the target protein.

以下、実施例を用いて本発明をより詳細に説明するが、本発明の技術的範囲はこれら実施例に限定されるものではない。
〔実施例1〕本発明に係る植物体栽培装置におけるミラクリン遺伝子組換えトマトを用いたミラクリン生産
1.植物体栽培装置
植物体栽培装置を、室内に温度及び二酸化炭素濃度の調節が可能な3.1m×4.5m×2.4mのプレハブ内に設置した。当該栽培装置は、幅94cm奥行き86cm高さ82cmの栽培室を2段積み重ねた構造を有し、4室の栽培室で1つの栽培装置とした。
EXAMPLES Hereinafter, although this invention is demonstrated in detail using an Example, the technical scope of this invention is not limited to these Examples.
[Example 1] Production of miraculin using miraculin transgenic tomatoes in the plant cultivation apparatus according to the present invention
1. Plant cultivating device The plant cultivating device was installed in a 3.1m x 4.5m x 2.4m prefab where the temperature and carbon dioxide concentration could be adjusted indoors. The cultivation apparatus has a structure in which cultivation rooms having a width of 94 cm, a depth of 86 cm, and a height of 82 cm are stacked in two stages, and four cultivation rooms constitute one cultivation apparatus.

各栽培室には上部にインバーターを付けた32W蛍光灯を12本配置し、さらに植物間照明装置として栽培用の樋と平行に24W蛍光灯3本を2カ所に配置した。栽培室正面奥には通風用のファンを3つ設置した。植物体栽培装置としては、改良型NFT方式を採用した。各栽培室に90cm×16cm×9cmの樋を3つ設置し、養液が循環できるように給液部と排液部を設けた。各樋には60Lの循環タンクから養液が供給され、植物が吸収できなかった養液は同じ循環タンクに回収されることとなる。循環タンク内の養液が不足した場合は、自動で調合された養液(大塚処方(EC=2.0dS/m))を貯蔵する養液タンクから循環タンクへと供給されるように設計した。   Each cultivation room was equipped with 12 32W fluorescent lamps with an inverter at the top, and three 24W fluorescent lamps were placed in two locations in parallel with the cultivation basket as an inter-plant lighting device. Three fans for ventilation were installed in the back of the cultivation room. The improved NFT method was adopted as the plant cultivation device. Three 90 cm x 16 cm x 9 cm cocoons were installed in each cultivation room, and a liquid supply unit and a drainage unit were provided so that the nutrient solution could circulate. The nutrient solution is supplied to each tub from a 60L circulation tank, and the nutrient solution that the plant could not absorb is collected in the same circulation tank. When the nutrient solution in the circulation tank is insufficient, the nutrient solution prepared automatically (Otsuka prescription (EC = 2.0 dS / m)) is designed to be supplied from the nutrient solution tank to the circulation tank.

2.材料及び方法
35Sプロモーターの制御下で恒常的にミラクリン遺伝子を発現する組換えトマト(導入品種Moneymaker)の固定済みT4世代種子を供試した(特許文献1)。当該種子を5cm×5cm×5cmのロックウールに播種し、インキュベーター内において250μmol/m2/sの光条件下で育苗した。
2.Materials and methods
A fixed T4 generation seed of recombinant tomato (introduced variety Moneymaker) that constantly expresses the miraculin gene under the control of the 35S promoter was used (Patent Document 1). The seeds were sown on 5 cm × 5 cm × 5 cm rock wool and grown in an incubator under light conditions of 250 μmol / m 2 / s.

30日間の育苗後、得られた苗を、苗テラス(大洋興業(株))に移動し10日間さらに育苗した後、上記植物体栽培装置に定植した。定植後、一本仕立てとして整枝し、第1果房から上位3枚の本葉を残して摘心した。植物体栽培装置では、長日条件(20時間照明/4時間暗黒)と短日条件(16時間照明/8時間暗黒)に設定した。さらに、光強度を強光(500μmol/m2/s)と弱光(400μmol/m2/s)を設定し、各光条件を組み合わせて4処理区を設けた。気温は、全ての栽培条件で明期25℃暗期20℃とした。また、CO2濃度はいずれも1000ppmに維持した。 After seedling for 30 days, the obtained seedlings were transferred to a seedling terrace (Taiyo Kogyo Co., Ltd.), further seeded for 10 days, and then planted in the plant cultivation apparatus. After planting, the branches were tailored as a single tailoring, and the top three leaves were removed from the first fruit bunch. In the plant cultivation apparatus, long day conditions (20 hours illumination / 4 hours dark) and short days conditions (16 hours illumination / 8 hours dark) were set. Further, strong light (500 μmol / m 2 / s) and weak light (400 μmol / m 2 / s) were set as the light intensity, and four treatment zones were provided by combining each light condition. The temperature was set to 25 ° C for light season and 20 ° C for dark season in all cultivation conditions. The CO 2 concentration was maintained at 1000 ppm.

果実が緑熟及び赤熟になった段階でそれぞれ収穫し、正常果と異常果数を記録した上で、果実重量を計測した。また、果実内におけるミラクリン遺伝子の発現強度をReal time PCR法で測定した。加えて、ミラクリンタンパク質の蓄積の有無についてウエスタンブロット法を用いて確認した。   The fruits were harvested at the stage of green ripening and red ripening, and the number of normal fruits and abnormal fruits were recorded, and the fruit weight was measured. In addition, the expression intensity of miraculin gene in the fruit was measured by Real time PCR. In addition, the presence or absence of accumulation of miraculin protein was confirmed using Western blotting.

3.結果及び考察
結果を下記の表1及び図4〜7に示す。
3. Results and Discussion The results are shown in Table 1 below and FIGS.

Figure 0005637343
Figure 0005637343

正常果実数は、長日強光下で多く、尻腐れ果の数は短日弱光下で多くなった(表1及び図4)。   The number of normal fruits was large under long day strong light, and the number of buttocks rot was increased under short day low light (Table 1 and FIG. 4).

一方、日長に関係なく強光下では、収穫までの日数は少なくなることに加えて、1株当たりの収量も緑熟果の場合は50%以上、赤熟果では40%以上、それぞれ多くなった(表1及び図5)。   On the other hand, under strong light regardless of the day length, the number of days until harvesting is reduced, and the yield per share is more than 50% for green ripe fruits and more than 40% for red ripe fruits. (Table 1 and FIG. 5).

図6は、果実内のミラクリン遺伝子の発現強度(ユビキチン遺伝子に対する相対発現強度)を示す。また、図7は、果実内におけるミラクリンタンパク質の蓄積の有無についてのウエスタンブロット結果を示す。なお、図7における「Pレーン」は、精製したミラクリンタンパク質サンプル(陽性対照)である。   FIG. 6 shows the expression intensity of the miraculin gene in fruits (relative expression intensity relative to the ubiquitin gene). Moreover, FIG. 7 shows the Western blot result about the presence or absence of the accumulation of miraculin protein in the fruit. “P lane” in FIG. 7 is a purified miraculin protein sample (positive control).

図6から判るように、ミラクリン遺伝子の発現強度は、緑熟、赤熟果実ともに長日強光下で他の栽培条件と比較して少なくなった。さらに、緑熟果実及び赤熟果実のミラクリンタンパク質の蓄積を確認すると、緑熟果実及び赤熟果実ともに長日強光下の果実ではバンドの強度にバラツキがあった(図7)。   As can be seen from FIG. 6, the expression intensity of the miraculin gene was reduced in both green and red ripened fruits under long day intense light compared to other cultivation conditions. Further, when the accumulation of miraculin protein in the green and red fruits was confirmed, both the green and red fruits had variations in band intensity in the fruits under long-day intense light (FIG. 7).

上述の植物体栽培装置を用いた栽培試験結果より、短日強光条件が最も効率よくミラクリン蓄積組換えトマトを生産できる光条件ではないかと考えられた。また、本実施例で設定した環境条件下において、育苗圃と本圃を分けて栽培する場合、年間5回程度の収穫を行うことが可能となり、1回収穫の場合には約5kg/m2/年、5回収穫の場合には約25 kg/m2/年のトマト収量となる試算結果を得た。また、育苗期間等の短縮を図れば6回収穫で約30 kg/m2/年のトマト収量も可能である。加えて、目的タンパク質収量にはその熟度が影響しないことが判明している。そこで、遺伝子組換えトマトにおける導入遺伝子によりコードされる目的タンパク質の生産を意図した場合には、成熟前果実を収穫する前提とした栽培期間の短縮により、最終的には40 kg/m2/年の遺伝子組換えトマト収量も可能である。 From the results of the above-described cultivation test using the plant cultivation apparatus, it was considered that the short-day intense light condition is the most efficient light condition for producing the miraculin-accumulating recombinant tomato. In addition, under the environmental conditions set in this example, when growing the seedling farm and the main farm separately, it is possible to harvest about five times a year, and in the case of one harvest, about 5 kg / m 2 / In the case of harvesting five times a year, a trial calculation result that yields a tomato yield of about 25 kg / m 2 / year was obtained. In addition, if the seedling period is shortened, a tomato yield of about 30 kg / m 2 / year is possible after six harvests. In addition, it has been found that the maturity does not affect the target protein yield. Therefore, when production of the target protein encoded by the transgene in genetically modified tomatoes is intended, the final cultivation period is 40 kg / m 2 / year by shortening the cultivation period on the premise of harvesting pre-ripe fruits. The genetically modified tomato yield is also possible.

さらに、本実施例により、ミラクリンタンパク質の蓄積量が長日強光下以外では安定していることが明らかとなった。   Furthermore, this example revealed that the accumulated amount of miraculin protein was stable except under long-day intense light.

本発明に係る植物体栽培装置の概略構成図である。It is a schematic block diagram of the plant cultivation apparatus concerning this invention. 本発明に係る植物体栽培装置の正面図及び側面図である。It is the front view and side view of a plant cultivation apparatus which concern on this invention. 本発明に係る植物体栽培装置における養液循環手段を示す図である。It is a figure which shows the nutrient solution circulation means in the plant cultivation apparatus which concerns on this invention. トマト果実の品質に及ぼす光環境の影響を示すグラフである。It is a graph which shows the influence of the light environment on the quality of a tomato fruit. トマト果実の総重量に及ぼす光環境の影響を示すグラフである。It is a graph which shows the influence of the light environment on the total weight of a tomato fruit. トマト果実内のミラクリン遺伝子の発現強度を示すグラフである。It is a graph which shows the expression intensity of the miraculin gene in a tomato fruit. トマト果実内のミラクリンタンパク質の蓄積の有無についてのウエスタンブロット結果を示す写真である。It is a photograph which shows the western blotting result about the presence or absence of accumulation of miraculin protein in tomato fruit.

符号の説明Explanation of symbols

1.植物体栽培装置、2.植物体、3.光環境調節手段、4.排気手段、5.水耕栽培手段、6.栽培室、7.循環タンク、8.養液自動調合装置、9.養液ストック、10.給液、11.排液、12.養液タンク、13.水道水 1. 1. Plant cultivation apparatus, 2. plant body; 3. Light environment adjusting means; 4. exhaust means; Hydroponic cultivation means, 6. 6. Cultivation room Circulation tank, 8. 8. Nutrient solution automatic preparation device, 9. Nutrient stock, 10. Liquid supply, 11. Drainage, 12. Nutrient solution tank, 13. Tap water

Claims (1)

遺伝子組換えトマト植物体に対する光環境を調節する光環境調節手段と、
光環境調節手段からの熱を調節する排気手段と、
薄膜水耕栽培手段と、
薄膜水耕栽培手段に対して養液を供給及び回収する養液循環手段と、
室内大気中炭酸ガス濃度調節手段と、
を備える栽培室を積層構造で複数備え、
前記光環境調節手段を前記遺伝子組換えトマト植物体に対して上面及び側面の双方に配置し、
前記遺伝子組換えトマト植物体は、ミラクリンをコードする遺伝子を有する遺伝子組換えトマト植物体であり、明期16〜20時間未満、光合成有効光量子束密度に基づく光強度300〜400μmol/m2/s及び炭酸ガス濃度500〜1000ppmを含む該遺伝子組換えトマト植物体栽培条件を制御するための遺伝子組換えトマト植物体栽培装置。
A light environment adjusting means for adjusting the light environment for the transgenic tomato plant,
An exhaust means for adjusting heat from the light environment adjusting means;
Thin film hydroponics,
A nutrient solution circulation means for supplying and recovering the nutrient solution to the thin-film hydroponics means;
Means for adjusting carbon dioxide concentration in the indoor atmosphere;
A plurality of cultivation rooms equipped with a laminated structure,
The light environment control means is disposed on both the upper surface and the side surface of the genetically modified tomato plant,
The genetically modified tomato plant is a genetically modified tomato plant having a gene encoding miraculin, and has a light period of 16 to less than 20 hours and a light intensity based on a photosynthetic effective photon flux density of 300 to 400 μmol / m 2 / A genetically modified tomato plant cultivating apparatus for controlling the genetically modified tomato plant cultivating conditions comprising s and a carbon dioxide gas concentration of 500 to 1000 ppm.
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