JP2022061807A - Method for introduction of material to pollen - Google Patents
Method for introduction of material to pollen Download PDFInfo
- Publication number
- JP2022061807A JP2022061807A JP2020169978A JP2020169978A JP2022061807A JP 2022061807 A JP2022061807 A JP 2022061807A JP 2020169978 A JP2020169978 A JP 2020169978A JP 2020169978 A JP2020169978 A JP 2020169978A JP 2022061807 A JP2022061807 A JP 2022061807A
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- Prior art keywords
- pollen
- hydrophilic
- substance
- support
- particle bombardment
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Abstract
Description
本発明は、花粉への物質導入方法に関し、より詳しくは、パーティクルボンバードメント法による花粉への物質導入方法に関する。 The present invention relates to a method for introducing a substance into pollen, and more particularly to a method for introducing a substance into pollen by a particle bombardment method.
核酸、タンパク質等の物質を、細胞内に導入することは、基礎研究のみならず、様々な産業用途においても非常に意義のあることである。しかしながら、植物は固い細胞壁を持ち、動物等に比べ、外部から物質を導入するのが困難である。このような、植物細胞への物質導入に関し、細胞壁を取り除いたプロトプラストの貪食作用を利用する方法（ポリエチレングリコール法）、微細なシリンジを用いて細胞内に物質を導入する方法（マイクロインジェクション法）、電気的に細胞膜に孔を開け、物質を細胞内に流入させる方法（エレクトロポレーション法）、微粒子を物質で覆って、それを細胞内に撃ち込む方法（パーティクルボンバードメント法）が試みられている。このうち、パーティクルボンバードメント法は、簡便で広範な植物に適用可能なことから、植物細胞への物質導入方法として、一般的に用いられている（非特許文献１）。しかも、この方法は、特に固い細胞壁を持つ成熟花粉等に有効であることが、様々な被子植物で報告されている（非特許文献２）。 Introducing substances such as nucleic acids and proteins into cells is very significant not only in basic research but also in various industrial applications. However, plants have a hard cell wall, and it is more difficult to introduce substances from the outside than animals and the like. Regarding the introduction of substances into plant cells, a method of utilizing the phagocytic action of protoplasts from which the cell wall has been removed (polyethylene glycol method), a method of introducing substances into cells using a fine syringe (microinjection method), Attempts have been made to electrically make holes in cell membranes and allow substances to flow into cells (electroporation method), and to cover fine particles with substances and shoot them into cells (particle bombardment method). Of these, the particle bombardment method is generally used as a method for introducing a substance into plant cells because it is simple and applicable to a wide range of plants (Non-Patent Document 1). Moreover, it has been reported in various angiosperms that this method is particularly effective for mature pollen having a hard cell wall (Non-Patent Document 2).
パーティクルボンバードメント法では、通常、花粉管伸長（培養）用の液体培地等に成熟花粉を懸濁し、花粉に吸水させた状態で、バキュームポンプで吸引しながらろ紙やナイロンメンブレン等の支持体上に花粉を集約し、導入する方法が主に用いられている（非特許文献３、非特許文献４）。また、寒天培地を支持体として用いる方法も用いられている（非特許文献５、６）。
In the particle bombardment method, mature pollen is usually suspended in a liquid medium for pollen tube elongation (culture), and the pollen absorbs water, and while sucking with a vacuum pump, it is placed on a support such as filter paper or nylon membrane. A method of collecting and introducing pollen is mainly used (
しかしながら、これらを支持体として用いたパーティクルボンバードメント法では、花粉への物質導入効率が低く、また正常に花粉管を発芽しない花粉も多い（すなわち導入処理後の生存率が低い）という問題点があった。 However, the particle bombardment method using these as a support has the problems that the efficiency of introducing substances into pollen is low and that many pollens do not germinate pollen tubes normally (that is, the survival rate after introduction treatment is low). there were.
本発明は、前記従来技術の有する課題に鑑みてなされたものであり、導入効率及び生存率高く、花粉に物質を導入することが可能なパーティクルボンバードメント法を提供することを目的とする。 The present invention has been made in view of the problems of the prior art, and an object of the present invention is to provide a particle bombardment method capable of introducing a substance into pollen with high introduction efficiency and survival rate.
本発明者らは、前記目的を達成すべく、様々な人工膜を、花粉を載せるための支持体として用い、当該花粉に対し、パーティクルボンバードメント法による物質導入を行なった。その結果、親水性ポリテトラフルオロエチレン（親水性ＰＴＦＥ）又は親水性混合セルロースエステル（親水性ＭＣＥ、すなわち、ニトロセルロース及びセルロースアセテートの混合物）を支持体として用いた場合には、物質が導入され、かつ生存して花粉管を発芽している花粉数が、他の支持体よりも顕著に多くなるということが明らかになった。 In order to achieve the above object, the present inventors used various artificial membranes as a support for mounting pollen, and introduced a substance into the pollen by a particle bombardment method. As a result, when hydrophilic polytetrafluoroethylene (hydrophilic PTFE) or hydrophilic mixed cellulose ester (hydrophilic MCE, that is, a mixture of nitrocellulose and cellulose acetate) is used as a support, the substance is introduced. Moreover, it was revealed that the number of pollen that survived and sprouted in the pollen tube was significantly higher than that of other supports.
一方、ＰＴＦＥであっても、疎水性ＰＴＦＥを支持体として用いた場合は、親水性のそれよりも、前記花粉数は少なかった。また、ニトロセルロース又はセルロースアセテートのみを支持体として用いた場合は、それらによって構成される親水性ＭＣＥよりも、前記花粉数は顕著に少なかった。また、従前より支持体として用いられている、ろ紙、ナイロン、寒天培地も、親水性ＰＴＦＥ又は親水性ＭＣＥと比して、物質が導入され、かつ生存して花粉管を発芽している花粉数は少なかった。さらに、親水性ＰＴＦＥを支持体として用いた場合に、花粉の中にある雄原細胞にまで、高い効率と生存率にて物質を導入できることを見出した。 On the other hand, even in the case of PTFE, when hydrophobic PTFE was used as a support, the number of pollen was smaller than that of hydrophilic one. Moreover, when only nitrocellulose or cellulose acetate was used as a support, the number of pollens was significantly smaller than that of the hydrophilic MCE composed of them. In addition, filter paper, nylon, and agar medium, which have been used as supports for some time, also have the number of pollen in which the substance is introduced and the pollen tube is germinated alive, as compared with the hydrophilic PTFE or the hydrophilic MCE. Was few. Furthermore, it has been found that when hydrophilic PTFE is used as a support, the substance can be introduced into male progenitor cells in pollen with high efficiency and survival rate.
すなわち、本発明は、花粉への物質導入方法に関し、より詳しくは、以下に記載の、パーティクルボンバードメント法による花粉への物質導入方法を提供する。
＜１＞ 花粉に物質を導入する方法であって、
パーティクルボンバードメント法により、物質で被覆した微粒子を支持体に載せた花粉に撃ち込む工程を含み、かつ
前記支持体が、親水性ポリテトラフルオロエチレン及び親水性混合セルロースエステルのうちの少なくとも１の高分子を含む支持体である、方法。
＜２＞ 前記物質が、ヌクレオチド及び／又はペプチドである、＜１＞に記載の方法。
＜３＞ 前記微粒子が金属微粒子である、＜１＞又は＜２＞に記載の方法。
＜４＞ 親水性ポリテトラフルオロエチレン及び親水性混合セルロースエステルのうちの少なくとも１の高分子を含む、パーティクルボンバードメント用花粉支持材。
＜５＞ ＜４＞に記載のパーティクルボンバードメント用花粉支持材を備える、パーティクルボンバードメント用装置。
That is, the present invention relates to a method for introducing a substance into pollen, and more particularly, provides a method for introducing a substance into pollen by the particle bombardment method described below.
<1> A method of introducing a substance into pollen.
By the particle bombardment method, a step of shooting fine particles coated with a substance into pollen placed on a support is included, and the support is a polymer of at least one of hydrophilic polytetrafluoroethylene and hydrophilic mixed cellulose ester. A support, including a method.
<2> The method according to <1>, wherein the substance is a nucleotide and / or a peptide.
<3> The method according to <1> or <2>, wherein the fine particles are metal fine particles.
<4> A pollen support material for particle bombardment, which comprises at least one polymer of hydrophilic polytetrafluoroethylene and hydrophilic mixed cellulose ester.
<5> A device for particle bombardment provided with the pollen support material for particle bombardment according to <4>.
本発明によれば、導入効率及び生存率高く、花粉に物質を導入することが可能となる。 According to the present invention, it is possible to introduce a substance into pollen with high introduction efficiency and survival rate.
本発明の花粉に物質を導入する方法は、パーティクルボンバードメント法により、物質で被覆した微粒子を支持体に載せた花粉に撃ち込む工程を含み、かつ前記支持体が、親水性ポリテトラフルオロエチレン及び親水性混合セルロースエステルのうちの少なくとも１の高分子を含む支持体である、方法である。 The method for introducing a substance into the pollen of the present invention includes a step of shooting fine particles coated with the substance into the pollen placed on a support by a particle bombardment method, and the support is made of hydrophilic polytetrafluoroethylene and hydrophilic. A method that is a support comprising at least one polymer of sex-mixed cellulose esters.
本発明において、「花粉」は、植物の雄性配偶子を意味し、成熟花粉のみならず、未成熟花粉も含まれる。未成熟花粉には、花粉母細胞、四分子（花粉四分子）、小胞子、２核の未熟花粉も含まれる。また、花粉を構成する細胞（雄原細胞、精細胞、花粉管細胞（栄養細胞）等）も、本発明の物質導入の対象となる花粉に含まれる。花粉の由来となる「植物」については特に制限はなく、例えば、双子葉植物及び単子葉植物を含む被子植物、裸子植物、草本植物、並びに木本植物が挙げられる。植物のより具体的な例としては、トマト、タバコ、ピーマン、トウガラシ、ナス等のナス類；キュウリ、カボチャ、スイカ等のウリ類；キャベツ、ブロッコリー、ハクサイ、シロイヌナズナ等の菜類；セルリ一、パセリ一、レタス等の生菜・香辛菜類；ネギ、タマネギ、ニンニク等のネギ類；ダイズ、ラッカセイ、インゲン、エンドウ、アズキ、リョクトウ、ササゲ、ソラマメ等の豆類；イチゴ、メロン等のその他果菜類；ダイコン、カブ、ニンジン、ゴボウ等の直根類；サトイモ、キャッサバ、ジャガイモ、サツマイモ、ナガイモ等のイモ類；イネ、トウモロコシ、コムギ、ソルガム、オオムギ、ライムギ、ミナトカモジグサ、ソバ等の穀類；アスパラガス、ホウレンソウ、ミツバ等の柔菜類；ユリ、トルコギキョウ、ストック、カーネーション、キク等の花弁類；ベントグラス、コウライシバ等の芝類；ナタネ、ラッカセイ、セイヨウアブラナ、ナンヨウアブラギリ等の油料作物類；ワ夕、イグサ等の繊維料作物類；クローバー、デントコーン、タルウマゴヤシ等の飼料作物類；リンゴ、ナシ、ブドウ、モモ、キウイフルーツ等の落葉性果樹類；ウンシュウミカン、オレンジ、レモン、グレープフルーツ等の柑橘類；サツキ、ツツジ、スギ、ポプラ、パラゴムノキ、イチョウ、マツ等の木本類等が挙げられる。 In the present invention, "pollen" means a male gamete of a plant, and includes not only mature pollen but also immature pollen. Immature pollen also includes pollen mother cells, four molecules (four pollen molecules), microspores, and two-nucleus immature pollen. In addition, cells constituting pollen (male progenitor cells, sperm cells, pollen tube cells (nutrient cells), etc.) are also included in the pollen to which the substance of the present invention is introduced. The "plant" from which the pollen is derived is not particularly limited, and examples thereof include angiosperms including dicotyledonous plants and monocotyledonous plants, angiosperms, herbaceous plants, and woody plants. More specific examples of plants include legumes such as tomatoes, tobacco, peppers, corn and eggplants; melons such as cucumbers, pumpkins and watermelons; vegetables such as turnips, broccoli, Chinese cabbage and azuki beans; 1. Raw vegetables and spicy vegetables such as lettuce; Negi such as green onions, onions and garlic; Beans such as soybean, Chinese cabbage, green beans, pea, azuki bean, ryokuto, potato and corn; Other fruit vegetables such as strawberry and melon; , Turnips, carrots, gobos, etc .; potatoes, cassaba, potatoes, sweet potatoes, potatoes, etc. , Mitsuba and other soft vegetables; Yuri, Turkish ginkgo, stock, carnation, Kiku and other petals; Bentgrass, corn and other turf; Textile crops; Forage crops such as clover, dent corn, and tarumago palm; deciduous fruit trees such as apples, pears, grapes, peaches, and kiwi fruits; Chinese cabbage such as unshu mikan, orange, lemon, and grapefruit; , Sugi, poplar, potatoes, ginkgo, pine and other woody plants.
前記の花粉に導入される「物質」としては特に制限はなく、例えば、ヌクレオチド（ＤＮＡ、ＲＮＡ）、ペプチド、糖、脂質等の生体高分子、蛍光色素等の低分子化合物が挙げられる。ヌクレオチドには、オリゴヌクレオチド、ポリヌクレオチド及び核酸が含まれ、ペプチドには、オリゴペプチド、ポリペプチド及びタンパク質が含まれ、糖には、オリゴ糖及び糖鎖が含まれる。また、本発明に係る「生体高分子」には、天然に存在するもののみならず、それらの誘導体（例えば、架橋型ヌクレオチド、非天然型アミノ酸）も含まれ、さらにそれらの複合体（例えば、糖タンパク質、糖脂質、ＲＮＡ－タンパク質複合体）も含まれる。 The "substance" introduced into the pollen is not particularly limited, and examples thereof include biopolymers such as nucleotides (DNA, RNA), peptides, sugars and lipids, and small molecule compounds such as fluorescent dyes. Nucleotides include oligonucleotides, polynucleotides and nucleic acids, peptides include oligopeptides, polypeptides and proteins, and sugars include oligosaccharides and sugar chains. Further, the "biopolymer" according to the present invention includes not only naturally occurring proteins but also derivatives thereof (for example, crosslinked nucleotides and unnatural amino acids), and further complex thereof (for example, for example). Glycoproteins, glycolipids, RNA-protein complexes) are also included.
本発明の花粉への物質導入方法においては、上記のとおり、パーティクルボンバードメント法により、前記物質で被覆した微粒子を支持体に載せた対象（本発明においては、花粉）に撃ち込む方法が用いられる。なお、「パーティクルボンバードメント法」は、パーティクルガン法、遺伝子銃法、微粒子銃法、微粒子射出法、パーティクルデリバリー法とも称される方法であり、ＤＮＡ等の物質で被覆した微粒子を高速で射出して対象の細胞内に導入する方法を意味する。以下、図１Ａ及び１Ｂを参照しながら本発明のパーティクルボンバードメント法の実施態様の例について説明する。 In the method for introducing a substance into pollen of the present invention, as described above, a method of shooting fine particles coated with the substance into an object (pollen in the present invention) placed on a support is used by the particle bombardment method. The "particle bombardment method" is also called a particle gun method, a gene gun method, a fine particle gun method, a fine particle ejection method, or a particle delivery method, and ejects fine particles coated with a substance such as DNA at high speed. It means a method of introducing into a target cell. Hereinafter, an example of an embodiment of the particle bombardment method of the present invention will be described with reference to FIGS. 1A and 1B.
パーティクルボンバードメント法においては、例えば、物質を撃ち込む前に、パーティクルボンバードメント装置１内を陰圧にした上で、ガス加速管２にガスをラプチャーディスク３が破壊圧力に達するまで供給される。次いで、図１Ｂに示すとおり、ラプチャーディスク３が破裂すること（破裂後のラプチャーディスク３ａ及び３ｂ）によって発生するガス衝撃圧力により、前記物質で被覆した微粒子５が下面に付着しているマクロキャリア４が、対象（花粉７）の方向に移動することとなる。そして、マクロキャリア４がストッピングスクリーン６で停止し、付着していた前記微粒子５のみが、支持体８に載せた対象（花粉７）へ撃ち込まれることとなる。
In the particle bombardment method, for example, before shooting a substance, the inside of the
本発明のパーティクルボンバードメント法において、花粉を載せるための支持体（「パーティクルボンバードメント用花粉支持材」とも称する）は、後記の実施例において示すとおり、花粉への物質の導入効率、及び該導入後の花粉の生存率が高いという観点から、親水性ポリテトラフルオロエチレン及び親水性混合セルロースエステルのうちの少なくとも１の高分子を含む支持体である。 In the particle bombardment method of the present invention, the support for mounting pollen (also referred to as "pollen support material for particle bombardment") has the efficiency of introducing a substance into pollen and its introduction, as shown in the examples below. From the viewpoint of high survival rate of pollen later, it is a support containing at least one polymer of hydrophilic polytetrafluoroethylene and hydrophilic mixed cellulose ester.
本発明において、「親水性」とは、水接触角度が９０度未満であることを意味する（なお、水接触角度とは、大気中、室温における水滴に対する静的接触角度のことである）。「親水性ポリテトラフルオロエチレン」は、疎水性であるポリテトラフルオロエチレン（ＰＴＦＥ）に親水処理を施すことによって調製される。「親水処理」としては、特に制限はなく、例えば、水酸基等の置換基を導入する方法、ポリビニルアルコール（ＰＶＡ）等の親水性樹脂を含浸させた後、これを架橋する方法、膜の表面にヒドロキシプロピルアクリル酸、テトラエチレングリコールアクリル酸、ポリエチレングリコールジアクリレート等で表面処理する方法、メタノール、エタノール、２－プロパノール等のアルコール類又はその他の表面張力の低い液体に浸漬する方法、電子線等のエネルギー線を照射することにより改質する方法、アルカリ金属材料による表面改質法が挙げられる。「親水性混合セルロースエステル（親水性ＭＣＥ）」は、ニトロセルロースとセルロースアセテートとの混合物であり、そもそも親水性である。 In the present invention, "hydrophilic" means that the water contact angle is less than 90 degrees (note that the water contact angle is a static contact angle with respect to water droplets in the air or at room temperature). "Hydrophilic polytetrafluoroethylene" is prepared by subjecting hydrophobic polytetrafluoroethylene (PTFE) to a hydrophilic treatment. The "hydrophilic treatment" is not particularly limited, and for example, a method of introducing a substituent such as a hydroxyl group, a method of impregnating a hydrophilic resin such as polyvinyl alcohol (PVA) and then cross-linking the same, and a method of cross-linking the surface of the film. Surface treatment with hydroxypropylacrylic acid, tetraethylene glycol acrylic acid, polyethylene glycol diacrylate, etc., immersion in alcohols such as methanol, ethanol, 2-propanol or other liquids with low surface tension, electron beam, etc. Examples thereof include a method of modifying by irradiating with energy rays and a surface modifying method using an alkali metal material. "Hydrophilic mixed cellulose ester (hydrophilic MCE)" is a mixture of nitrocellulose and cellulose acetate, and is hydrophilic in the first place.
これら親水性高分子は、当業者であれば公知の製造方法により適宜調製することができる。また、多々市販もされているので、それらを購入することによっても用意することができる。例えば、親水性ＰＴＦＥは、ＭＥＲＣＫ社から「製品名：Ｏｍｎｉｐｏｒｅ（登録商標）」が市販されており、ＡＤＶＡＮＴＥＣ社から「製品名：親水性ＰＴＦＥメンブレンフィルタ」が市販されている。親水性ＭＣＥは、ＭＥＲＣＫ社から「製品名：ＭＦ－Ｍｉｌｌｉｐｏｒｅ（登録商標）」が市販されており、ＡＤＶＡＮＴＥＣ社から「製品名：セルロース混合エステルタイプ メンブレンフィルター」が市販されており、Ｃｙｔｉｖａ社（旧：ＧＥヘルスケア社）から「製品名：セルロース混合エステルメンブレン」が市販されている。 These hydrophilic polymers can be appropriately prepared by those skilled in the art by a known production method. Also, since many are commercially available, they can be prepared by purchasing them. For example, as for hydrophilic PTFE, "Product name: Omnipore (registered trademark)" is commercially available from MERCK, and "Product name: Hydrophilic PTFE membrane filter" is commercially available from ADVANTEC. As for hydrophilic MCE, "Product name: MF-Millipore (registered trademark)" is commercially available from MERCK, and "Product name: Cellulose mixed ester type membrane filter" is commercially available from ADVANTEC, and Cytiva (formerly). : GE Healthcare), "Product name: Cellulose mixed ester membrane" is commercially available.
本発明に係る「支持体」は、緻密質であっても、多孔質であってもよいが、花粉が発芽するのに適した水分量を安定的に保つ観点から、多孔質であることが好ましい。「支持体」の形状は、特に制限されず、メンブレン状、フィルム状、シート状、板状であってもよいが、花粉の置床や、ボンバードメント後の培養が容易であるといった観点から、メンブレン状であることが好ましい。また、その形状にもよるが、図１Ａに例示するように、花粉は通常、支持体の一方の表面上に載せられる。本発明に係る「支持体」としては、花粉を載せるその表面の少なくとも一部が親水性ＰＴＦＥ及び／又は親水性ＭＣＥとなっていることが望ましく、他の素材を含んでいてもよい。ここで「一部」としては、支持体の一方の全表面の５０％以上、好ましくは７０％以上、より好ましくは８５％以上である。また、かかる他の素材としては、親水性ＰＴＦＥ及び／又は親水性ＭＣＥを裏打ちするための、プラスチック板（材質：ポリスチレン等）、親水性ＭＣＥ及び親水性ＰＴＦＥ以外の高分子からなるメンブレン（例えば、表１～３に記載のメンブレン）、ろ紙、寒天培地、ガラス板、金属板（アルミ板、ステンレス板等）、シリコーン樹脂（ＰＤＭＳ等）等が挙げられる。 The "support" according to the present invention may be dense or porous, but may be porous from the viewpoint of stably maintaining a water content suitable for pollen germination. preferable. The shape of the "support" is not particularly limited and may be membrane-like, film-like, sheet-like, or plate-like, but from the viewpoint of easy placement of pollen and culturing after bombardment, the membrane is used. It is preferably in the form of a shape. Also, depending on its shape, as illustrated in FIG. 1A, pollen is usually placed on one surface of the support. As the "support" according to the present invention, it is desirable that at least a part of the surface on which pollen is placed is hydrophilic PTFE and / or hydrophilic MCE, and other materials may be contained. Here, the "part" is 50% or more, preferably 70% or more, and more preferably 85% or more of the entire surface of one of the supports. Further, as such other materials, a plastic plate (material: polystyrene or the like) for lining the hydrophilic PTFE and / or the hydrophilic MCE, a membrane made of a polymer other than the hydrophilic MCE and the hydrophilic PTFE (for example, for example). The membranes shown in Tables 1 to 3), filter paper, agar medium, glass plate, metal plate (aluminum plate, stainless steel plate, etc.), silicone resin (PDMS, etc.) and the like can be mentioned.
支持体が多孔質メンブレン状の親水性ＰＴＦＥ及び／又は親水性ＭＣＥを含む場合には、親水性ＰＴＦＥ及び／又は親水性ＭＣＥの孔径が、通常０．１～１００μｍであり、好ましくは０．２～５０μｍであり、より好ましくは０．８～１０μｍである。さらに、支持体における親水性ＰＴＦＥ及び／又は親水性ＭＣＥの厚さは、通常１０～５００μｍであり、好ましくは１５～３００μｍであり、より好ましくは１９～１５０μｍである。 When the support contains a porous membrane-like hydrophilic PTFE and / or a hydrophilic MCE, the pore size of the hydrophilic PTFE and / or the hydrophilic MCE is usually 0.1 to 100 μm, preferably 0.2. It is ~ 50 μm, more preferably 0.8-10 μm. Further, the thickness of the hydrophilic PTFE and / or the hydrophilic MCE in the support is usually 10 to 500 μm, preferably 15 to 300 μm, and more preferably 19 to 150 μm.
また、花粉の生存に適した水分量を安定的に保つ観点から、支持体において親水性ＰＴＦＥ及び／又は親水性ＭＣＥは、液体を含んでいることが望ましい。かかる液体としては、例えば、花粉維持用培地（花粉管伸長培地、雄性配偶子初期培地等）、生理食塩水、リン酸緩衝液（ＰＢＳ）等の緩衝液が挙げられる。また、液体の含有率は、対象（花粉）の種類等によって当業者であれば適宜調整され得、特に制限されないが、例えば、０．０１～１０μＬ／ｍｍ３（１～３μＬ／ｍｍ３等）が挙げられる。 Further, from the viewpoint of stably maintaining a water content suitable for pollen survival, it is desirable that the hydrophilic PTFE and / or the hydrophilic MCE in the support contains a liquid. Examples of such a liquid include a pollen maintenance medium (pollen tube extension medium, male gamete initial medium, etc.), physiological saline, and a buffer solution such as phosphate buffer (PBS). The liquid content may be appropriately adjusted by those skilled in the art depending on the type of the target (pollen) and the like, and is not particularly limited, but is, for example, 0.01 to 10 μL / mm 3 (1 to 3 μL / mm 3 etc.). Can be mentioned.
本発明のパーティクルボンバードメント法において、前記物質によって被覆される「微粒子」の素材は、特に制限されず、例えば、金、タングステン、磁性粒子（四酸化三鉄等）等の金属からなる微粒子（金属微粒子）が挙げられる。これらの中でも、金粒子が好ましい。微粒子の粒子径は、特に制限されないが、例えば０．０５～５μｍであり、好ましくは０．１～３μｍであり、より好ましくは０．２～ｌ．５μｍであり、さらに好ましくは０．４～０．８μｍである。 In the particle bombardment method of the present invention, the material of the "fine particles" coated with the substance is not particularly limited, and for example, fine particles (metals) made of metal such as gold, tungsten, and magnetic particles (triiron tetroxide, etc.). Fine particles). Among these, gold particles are preferable. The particle size of the fine particles is not particularly limited, but is, for example, 0.05 to 5 μm, preferably 0.1 to 3 μm, and more preferably 0.2 to l. It is 5 μm, more preferably 0.4 to 0.8 μm.
本発明に係る「被覆」には、微粒子表面の全部が前記物質によって覆われていることのみならず、その一部が覆われている〈微粒子表面の一部に前記物質が付着している）状態も含まれる。ここで「一部」としては、微粒子表面の５０％以上、好ましくは８５％以上、より好ましくは９０％以上、さらに好ましくはほぼ全面（例えば、９５％以上）である。また「物質で被覆した微粒子」は、物質と微粒子との混合物の形態であってもよい。 In the "coating" according to the present invention, not only the entire surface of the fine particles is covered with the substance, but also a part thereof is covered (the substance is attached to a part of the surface of the fine particles). The state is also included. Here, the "part" is 50% or more, preferably 85% or more, more preferably 90% or more, and further preferably almost the entire surface (for example, 95% or more) of the surface of the fine particles. Further, the "fine particles coated with a substance" may be in the form of a mixture of the substance and the fine particles.
微粒子への前記物質の被覆方法は、特に制限されず、公知の方法に従った方法を採用することができる。例えば、微粒子と導入物を接触させることにより被覆することができる。この際に、プラスミドベクター等の立体構造解消のためにプラスチャージの物質（例えば、スペルミジン等のポリアミン）や、導入物の微粒子への付着や微粒子の沈殿を促進する物質（例えば、塩化カルシウム等の塩）を添加することによって、より効率的に被覆することができる。 The method for coating the fine particles with the substance is not particularly limited, and a method according to a known method can be adopted. For example, it can be coated by contacting the fine particles with the introduced material. At this time, a positively charged substance (for example, a polyamine such as spermidine) for eliminating the three-dimensional structure of a plasmid vector or the like, or a substance that promotes the adhesion of the introduced substance to the fine particles or the precipitation of the fine particles (for example, calcium chloride). By adding (salt), it can be coated more efficiently.
物質で被覆した微粒子は、通常、マイクロピペット等を用いてマクロキャリアに可能な限り均一に塗布した後、クリーンベンチ等の無菌環境中で乾燥させることによって、前記物質で被覆した微粒子が下面に付着しているマクロキャリアは調製される。 The fine particles coated with the substance are usually applied to the macrocarrier as uniformly as possible using a micropipette or the like, and then dried in a sterile environment such as a clean bench so that the fine particles coated with the substance adhere to the lower surface. The macrocarriers that are used are prepared.
前記物質で被覆した微粒子を撃ち込む方法は特に制限されないが、上記のとおり、通常、一定の圧力に設定されたガスにより射出する方法が採られる。ガスは、不活性ガスが好ましく用いられ、例えばヘリウムガスが採用される。 The method of shooting the fine particles coated with the substance is not particularly limited, but as described above, a method of injecting with a gas set to a constant pressure is usually adopted. As the gas, an inert gas is preferably used, and for example, helium gas is adopted.
ガス圧としては、好ましくは３０～２２００ｐｓｉ、より好ましくは４５０～１２００ｐｓｉである。なお、ガス圧は、微粒子の種類、対象（花粉）の種類、対象との距離等に応じて、一過的発現実験等により、適宜最適な圧力を決定することができる。 The gas pressure is preferably 30 to 2200 psi, more preferably 450 to 1200 psi. The optimum pressure of the gas pressure can be appropriately determined by a transient expression experiment or the like according to the type of fine particles, the type of the target (pollen), the distance from the target, and the like.
ラプチャーディスクとマクロキャリアとの距離としては、好ましくは２～１２ｃｍ、より好ましくは２～４ｃｍである。また、マクロキャリアと対象（花粉）との距離としては、好ましくは２～１２ｃｍ、より好ましくは３～６ｃｍである。なお、これらの距離は、微粒子の種類、対象（花粉）の種類等に応じて、一過的発現実験等により、適宜最適な圧力を決定することができる。 The distance between the rupture disc and the macro carrier is preferably 2 to 12 cm, more preferably 2 to 4 cm. The distance between the macro carrier and the target (pollen) is preferably 2 to 12 cm, more preferably 3 to 6 cm. The optimum pressure for these distances can be appropriately determined by a transient expression experiment or the like according to the type of fine particles, the type of target (pollen), and the like.
花粉に微粒子を撃ち込む回数としては、少なくとも１回（例えば、１回、２回）あれば良い。なお、撃ち込む回数についても、一過的発現実験等により、適宜最適な回数を決定することができる。 The number of times the fine particles are shot into the pollen may be at least once (for example, once or twice). As for the number of shots, the optimum number can be appropriately determined by a transient expression experiment or the like.
以上、本発明のパーティクルボンバードメント法の好適な実施形態について、図１Ａ及びＢに示した概略図に沿って説明したが、本発明は、当該図に示した形態のパーティクルボンバードメント装置に限定されることなく、物質を被覆した微粒子を高速で射出できる装置（所謂、遺伝子銃、パーティクルデリバリーシステム）であれば、その形態、射出形式等を問わず、利用することができる。より具体的には、チャンバー式、据え置き型、箱型又は設置型の遺伝子銃等（例えば、ＢＩＯ－ＲＡＤ社製 ＰＤＳ－１０００／Ｈｅシステム）に限定されることなく、ハンドヘルド型の遺伝子銃等（例えば、ＢＩＯ－ＲＡＤ社製 Ｈｅｌｉｏｓ Ｇｅｎｅ Ｇｕｎシステム）であっても、当業者であれば、適宜その装置に合わせた条件等を設定し、本発明を実施し得る。 Although the preferred embodiment of the particle bombardment method of the present invention has been described above with reference to the schematic views shown in FIGS. 1A and 1B, the present invention is limited to the particle bombardment apparatus of the form shown in the figure. Any device (so-called gene gun, particle delivery system) capable of injecting fine particles coated with a substance at high speed can be used regardless of its form, injection type, and the like. More specifically, it is not limited to chamber type, stationary type, box type or stationary type gene guns (for example, PDS-1000 / He system manufactured by BIO-RAD), and handheld type gene guns and the like (for example). For example, even in the case of a Helios Gene Gun system manufactured by BIO-RAD, a person skilled in the art can appropriately set conditions and the like suitable for the device and carry out the present invention.
以下、実施例に基づいて本発明をより具体的に説明するが、本発明は以下の実施例に限定されるものではない。 Hereinafter, the present invention will be described in more detail based on Examples, but the present invention is not limited to the following Examples.
（実施例１）
パーティクルボンバードメント法による花粉への物質導入において、適した花粉支持体を見出すべく、プラスミドＤＮＡを金の微粒子表面に被覆して、各種支持体に載せたベンサミアナタバコの花粉に撃ち込んだ。そして、プラスミドＤＮＡが導入され、生存していた花粉数を計測した。具体的には以下のようにして行なった。
(Example 1)
In the introduction of substances into pollen by the particle bombardment method, plasmid DNA was coated on the surface of fine gold particles and shot into the pollen of Nicotiana benthamiana mounted on various supports in order to find a suitable pollen support. Then, the plasmid DNA was introduced and the number of surviving pollens was counted. Specifically, it was carried out as follows.
＜プラスミドＤＮＡ＞
花粉に導入するプラスミドＤＮＡは、シロイヌナズナＵＢＱ１０（ポリユビキチン１０；ＡＴ４Ｇ０５３２０）遺伝子プロモーター下流にｓＧＦＰ遺伝子をつないだ配列（ＡｔＵＢＱ１０ｐ：：ｓＧＦＰ）を、ベンサミアナタバコ用のバイナリーベクター（Ａｄｄｇｅｎｅ社製、ｐＧｒｅｅｎ００２９）に挿入し、調製した。当該プラスミドＤＮＡが導入された花粉の細胞質（花粉及び花粉管）にて蛍光タンパク質（ＧＦＰ）が発現することになるので、これを指標として、プラスミドＤＮＡ導入花粉を判別することができる。
<Plasmid DNA>
The plasmid DNA to be introduced into pollen is a sequence (AtUBQ10p :: sGFP) in which the sGFP gene is linked downstream of the Arabidopsis thaliana UBQ10 (
＜プラスミドＤＮＡによる金粒子のコーティング＞
０．６μｍ径の金粒子（ＢＩＯ－ＲＡＤ社、＃１６５－２２６２）を１００％エタノールで洗浄し、滅菌水で懸濁して３０ｍｇ／ｍＬ金粒子含有水を調製した。後記のＤＮＡ被覆金粒子含有エタノールを１回の撃ち込みにつき１０μＬ使用することとし、当該エタノール中のプラスミドＤＮＡが１回の撃ち込みにつき５００ｎｇになるよう混合して加え、撹拌した。この金粒子及びプラスミドＤＮＡの混合液に対し、４μＬの０．１Ｍスペルミジンと１０μＬの２．５Ｍ塩化カルシウムとを撹拌しながら加え、スピンダウンによりプラスミドＤＮＡがコーティングされた金粒子を沈殿させた。その後、上清を除き、２０μＬの７０％エタノールで洗浄したのち、１０μＬの１００％エタノールで再懸濁し、ＤＮＡ被覆金粒子含有エタノールを調製した。
<Coating of gold particles with plasmid DNA>
Gold particles having a diameter of 0.6 μm (BIO-RAD, # 165-2262) were washed with 100% ethanol and suspended in sterile water to prepare water containing 30 mg / mL gold particles. It was decided to use 10 μL of ethanol containing DNA-coated gold particles described later per shot, and the plasmid DNA in the ethanol was mixed and added so as to be 500 ng per shot, and the mixture was stirred. 4 μL of 0.1 M spermidine and 10 μL of 2.5 M calcium chloride were added to the mixed solution of the gold particles and the plasmid DNA with stirring, and the gold particles coated with the plasmid DNA were precipitated by spin-down. Then, the supernatant was removed, and the mixture was washed with 20 μL of 70% ethanol and then resuspended with 10 μL of 100% ethanol to prepare ethanol coated with DNA-coated gold particles.
＜パーティクルボンバードメント法＞
パーティクルボンバードメントによる導入に用いるマクロキャリア１枚につき、前記ＤＮＡ被覆金粒子含有エタノールを１０μＬ使用した。当該エタノールをマクロキャリア上に滴下し、風乾した後に、導入装置（ＰＤＳ－１０００／Ｈｅ、ＢＩＯ－ＲＡＤ社、＃１６５－２２５７ Ｊ１）本体の最上段、すなわちラプチャーディスクからの距離が３ｃｍの位置となるようマクロキャリアを配置した。生育しているベンサミアナタバコの花から成熟花粉（葯３個分、開葯後の葯から採取した雄原細胞を持つ花粉）を採取し、各種支持体上に花粉を散布し、マクロキャリアからの距離が３ｃｍ又は６ｃｍの位置に配置した。
<Particle bombardment method>
10 μL of the DNA-coated gold particle-containing ethanol was used for each macro carrier used for introduction by particle bombardment. After dropping the ethanol onto the macro carrier and air-drying it, the top of the main body of the introduction device (PDS-1000 / He, BIO-RAD, # 165-2257 J1), that is, the position where the distance from the rupture disk is 3 cm. The macro carrier was arranged so as to be. Mature pollen (3 anthers, pollen with male progenitor cells collected from anthers after opening) is collected from the growing flowers of Nicotiana benthamiana, pollen is sprayed on various supports, and macrocarriers are used. It was placed at a position where the distance from was 3 cm or 6 cm.
支持体としては、下記表１～３に示す、花粉管伸長培地で湿らせた各種人工膜及びろ紙、並びに１％アガロースを加えた花粉管伸長培地を用いた。 As the support, various artificial membranes and filter papers moistened with the pollen tube extension medium shown in Tables 1 to 3 below, and the pollen tube extension medium containing 1% agarose were used.
なお、人工膜及びろ紙については、裏打ちするためにプラスチック板を敷いた。また、各種人工膜への単位体積あたりの培地滴下量は、１．３５μＬ／ｍｍ３とした。また、花粉管伸長培地の組成は以下のとおりである。
花粉管伸長培地の組成：０．０１ｗ／ｖ％ ホウ酸、１ｍＭ 塩化カルシウム、１ｍＭ 硝酸カルシウム、１ｍＭ 硫酸マグネシウム、１０ｗ／ｖ％ スクロース（水酸化カリウムにてｐＨ６．５に調整）（Ｈａｏ Ｗａｎｇ ＆ Ｌｉｗｅｎ Ｊｉａｎｇ、Ｎａｔｕｒｅ ｐｒｏｔｏｃｏｌｓ、２０１１年、６巻、４１９～４２６ページ 参照のほど）。
For the artificial membrane and filter paper, a plastic plate was laid to line them. The amount of medium dropped onto various artificial membranes per unit volume was 1.35 μL / mm 3 . The composition of the pollen tube extension medium is as follows.
Composition of pollen tube extension medium: 0.01 w / v% boric acid, 1 mM calcium chloride, 1 mM calcium nitrate, 1 mM magnesium sulfate, 10 w / v% sucrose (adjusted to pH 6.5 with potassium hydroxide) (Hao Wang & Liwen) Jiang, Nature protocols, 2011, Vol. 6, pp. 419-426).
１，１００ｐｓｉ用のラプチャーディスクを用い、ヘリウムガス圧１，１００ｐｓｉ，減圧－２５～２７ｉｎＨｇで１回ずつ撃ち込みを行なった。その後、人工膜上で撃ち込みを行なったものについては、人工膜ごと花粉を１％アガロースを加えた花粉管伸長培地上に移し、保湿、遮光して室温にて約２０時間培養した。 Using a rupture disc for 1,100 psi, the helium gas pressure was 1,100 psi, and the pressure was reduced to -25 to 27 inHg. Then, for those that had been shot on the artificial membrane, the pollen together with the artificial membrane was transferred onto a pollen tube elongation medium containing 1% agarose, moistened, shielded from light, and cultured at room temperature for about 20 hours.
そして、蛍光顕微鏡下で花粉及び花粉管のＧＦＰ蛍光の発現を観察した。蛍光タンパク質を発現している花粉管を、プラスミドＤＮＡが導入され、かつ生存して花粉管を発芽している花粉として計測し、支持体間にてその数を比較した。得られた結果を図２に示す。 Then, the expression of GFP fluorescence in pollen and pollen tubes was observed under a fluorescence microscope. Pollen tubes expressing the fluorescent protein were measured as pollen in which plasmid DNA was introduced and the pollen tubes were alive and germinated, and the numbers were compared between the supports. The obtained results are shown in FIG.
図２に示した結果から明らかなように、親水性ポリテトラフルオロエチレン（親水性ＰＴＦＥ）又は親水性混合セルロースエステル（親水性ＭＣＥ）を支持体として用いた場合には、プラスミドＤＮＡが導入され、かつ生存して花粉管を発芽している花粉数が、他の支持体よりも顕著に多かった（図２の１．、２．及び５． 参照）。 As is clear from the results shown in FIG. 2, when hydrophilic polytetrafluoroethylene (hydrophilic PTFE) or hydrophilic mixed cellulose ester (hydrophilic MCE) is used as a support, the plasmid DNA is introduced. Moreover, the number of pollen that survived and sprouted pollen tubes was significantly higher than that of other supports (see 1, 2, and 5 in FIG. 2).
一方、ＰＴＦＥであっても、疎水性ＰＴＦＥは、親水性のそれよりも、前記花粉数は少なかった（図２の３．及び４． 参照）。また、ニトロセルロース又はセルロースアセテートのみでは、それらによって構成される混合セルロースエステルよりも、前記花粉数は顕著に少なかった（図２の６．～９． 参照）。また、従前より支持体として用いられている、ろ紙、ナイロン、寒天培地（１％アガロース）も、親水性ＰＴＦＥ又は混合セルロースエステルと比して、プラスミドＤＮＡが導入され、かつ生存して花粉管を発芽している花粉数は少なかった。 On the other hand, even in the case of PTFE, the hydrophobic PTFE had a smaller number of pollen than the hydrophilic one (see 3 and 4 in FIG. 2). In addition, the number of pollen was significantly smaller with nitrocellulose or cellulose acetate alone than with the mixed cellulose ester composed of them (see 6 to 9 in FIG. 2). In addition, filter paper, nylon, and agar medium (1% agarose), which have been used as supports in the past, also have plasmid DNA introduced and survive pollen tubes as compared with hydrophilic PTFE or mixed cellulose ester. The number of sprouting pollen was small.
（実施例２）
実施例１にて花粉（細胞質）への導入数が一番良かった親水性ＰＴＦＥを用い、花粉内の雄原細胞に対しても効率良く物質を導入できるかにつき、上記ＡｔＵＢＱ１０ｐ：：ｓＧＦＰを有するプラスミドＤＮＡの代わりに、花粉の核を標識可能な下記３５Ｓｐ：：Ｈ２Ｂ－ｔｄＴｏｍａｔｏを有するプラスミドＤＮＡを用いた。
(Example 2)
Using the hydrophilic plasmid having the highest number of introductions into pollen (cytoplasm) in Example 1, the above-mentioned AtUBQ10p :: sGFP is used to determine whether the substance can be efficiently introduced into male progenitor cells in pollen. Instead of the plasmid DNA, a plasmid DNA having the following 35Sp :: H2B-tdTomato capable of labeling pollen nuclei was used.
＜プラスミドＤＮＡ＞
カリフラワーモザイクウイルス（ＣａＭＶ）の３５Ｓプロモーター下流に、シロイヌナズナヒストンＨ２Ｂ（ＡＴ１Ｇ０７７９０）由来の核移行シグナル及びｔｄＴｏｍａｔｏからなる融合タンパク質をコードする遺伝子をつないだ配列（３５Ｓｐ：：Ｈ２Ｂ－ｔｄＴｏｍａｔｏ）を、ＳｐＵＣベクターに挿入し、花粉に導入するプラスミドＤＮＡを調製した。当該プラスミドＤＮＡが導入された花粉の核（雄原細胞の核を含む）にて蛍光タンパク質（ｔｄＴｏｍａｔｏ）が発現することになる。
<Plasmid DNA>
A sequence (35Sp :: H2B-tdTomato) in which a gene encoding a fusion protein consisting of a nuclear translocation signal derived from white inunazuna histone H2B (AT1G07790) and a fusion protein consisting of tdTomato is linked downstream of the 35S promoter of cauliflower mosaic virus (CaMV) is used as a SpUC vector. The plasmid DNA that was inserted and introduced into the pollen was prepared. The fluorescent protein (tdTomato) will be expressed in the pollen nucleus (including the nucleus of the male progenitor cell) into which the plasmid DNA has been introduced.
さらに、支持体（親水性ＰＴＦＥ又は１％アガロース花粉管伸長培地）とマクロキャリアとの距離を３ｃｍのみにした以外は、実施例１同様に、パーティクルボンバードメントを行ない、蛍光タンパク質を発現している花粉管内の雄原細胞の数を計測し、支持体として、親水性ＰＴＦＥを用いた場合と、１％アガロース花粉管伸長培地を用いた場合とで比較した。得られた結果を図３に示す。 Further, particle bombardment was performed and the fluorescent protein was expressed in the same manner as in Example 1 except that the distance between the support (hydrophilic PTFE or 1% agarose pollen tube elongation medium) and the macrocarrier was only 3 cm. The number of male progenitor cells in the pollen tube was measured, and a comparison was made between the case where hydrophilic PTFE was used as a support and the case where 1% agarose pollen tube elongation medium was used. The obtained results are shown in FIG.
図３に示した結果から明らかなように、親水性ＰＴＦＥを支持体として用いた場合には、１％アガロースを用いた場合よりも、蛍光タンパク質を発現している花粉管内の雄原細胞の数は多かった。 As is clear from the results shown in FIG. 3, when hydrophilic PTFE was used as a support, the number of male progenitor cells in the pollen duct expressing the fluorescent protein was higher than that when 1% agarose was used. Was a lot.
（実施例３）
本発明の方法が、特定の植物種を対象とするものではないことを確認すべく、実施例１に記載と同様の方法にて、トルコギキョウの花粉への物質導入を試みた。
(Example 3)
In order to confirm that the method of the present invention does not target a specific plant species, an attempt was made to introduce a substance into pollen of Turkish ginkgo by the same method as described in Example 1.
具体的には、パーティクルボンバードメント法において、上記ベンサミアナタバコの花からの成熟花粉の代わりに、トルコギキョウの花からの成熟花粉（葯１個分、開葯後の葯から採取した雄原細胞を持つ花粉）を用いた。また、マクロキャリアと支持体との距離は３ｃｍに統一した。さらに、各種人工膜に添加した、花粉管伸長培地の組成を、以下に変更した。
１．６ｍＭ ホウ酸、１．８ｍＭ 硝酸カルシウム、２ｍＭ 酢酸、１５ｗ／ｖ％ スクロース（水酸化カリウムにてｐＨ５．７に調整）（Ｐａｒｋ Ｈｅｅ Ｓｕｎｇ、Ｊｏｕｒｎａｌ ｏｆ Ｌｉｆｅ Ｓｃｉｅｎｃｅ、２００４年、１４巻６号、１０１８～１０２２ページ．参照のほど）。
Specifically, in the particle bombardment method, instead of the mature pollen from the flower of Nicotiana benthamiana, the mature pollen from the flower of Turkish ginkgo (one anther, male progenitor cells collected from the anther after opening). Pollen with) was used. In addition, the distance between the macro carrier and the support was unified to 3 cm. Furthermore, the composition of the pollen tube elongation medium added to various artificial membranes was changed to the following.
1.6 mM boric acid, 1.8 mM calcium nitrate, 2 mM acetic acid, 15 w / v% sucrose (adjusted to pH 5.7 with potassium hydroxide) (Park Hee Sung, Journal of Life Science, 2004, Vol. 14, No. 6, Pages 1018-1022. See).
そして、以上の変更以外は、実施例１に記載の方法と同様にしてトルコギキョウの花粉への物質導入を試みた。得られた結果を表４に示す。 Then, except for the above changes, an attempt was made to introduce the substance into the pollen of Turkish ginkgo in the same manner as in the method described in Example 1. The results obtained are shown in Table 4.
表４に示した結果から明らかなように、トルコギキョウの花粉を対象としても、支持体に親水性ＰＴＦＥ又は親水性ＭＣＥを用いた場合の方が、従前より支持体として用いられている寒天培地（１％アガロース）と比較して、プラスミドＤＮＡが導入され、かつ生存して花粉管を発芽している花粉数は多かった（表４中の「蛍光」項目を参照のほど）。 As is clear from the results shown in Table 4, even when pollen of Turkish ginkgo is targeted, the agar medium (which has been used as a support in the past when hydrophilic plasmid or hydrophilic MCE is used as a support is used. Compared to 1% agarose), the number of pollen into which plasmid DNA was introduced and which survived and germinated pollen tubes was higher (see the “Fluorescent” item in Table 4).
したがって、本発明の方法によれば、植物の種類によらず、生存率及び導入効率高く、花粉に物質を導入できることが確認された。 Therefore, according to the method of the present invention, it was confirmed that the substance can be introduced into pollen with high survival rate and introduction efficiency regardless of the type of plant.
（実施例４）
本発明の方法が、成熟した花粉のみを対象とするものではないことを確認すべく、未成熟なステージにある雄性配偶子（四分子、小胞子、２核の未熟花粉）への物質導入を、以下に示す方法にて試みた。
(Example 4)
In order to confirm that the method of the present invention is not intended only for mature pollen, introduction of a substance into a male gamete (four molecules, microspores, two nuclei of immature pollen) at an immature stage is introduced. , I tried by the method shown below.
＜パーティクルボンバードメント法＞
実施例１に記載と同様にして、前記ＤＮＡ被覆金粒子が下面に付着しているマクロキャリアを調製し、導入装置において、ラプチャーディスクからの距離が３ｃｍの位置となるよう配置した。
<Particle bombardment method>
In the same manner as described in Example 1, a macro carrier to which the DNA-coated gold particles were attached to the lower surface was prepared and arranged in the introduction device so that the distance from the rupture disk was 3 cm.
物質導入の対象とする雄性配偶子のステージは以下のとおりであり、生育しているベンサミアナタバコの花からそれらの蕾の大きさを指標として調製した。
（１）四分子・小胞子が混在したステージ
（２）小胞子からなるステージ
（３）２核の未熟花粉からなるステージ
なお、（１）及び（２）においては、葯２０個分の雄性配偶子を採取した。（３）においては、葯１５個分の雄性配偶子を採取した。また、回収後、各雄性配偶子の一部を取り分け、ＤＡＰＩ染色することによって、雄性配偶子内の核の数を観察し、各ステージにあることを確認した。
The stages of male gametes targeted for substance introduction are as follows, and were prepared from the growing flowers of Nicotiana benthamiana using the size of their buds as an index.
(1) Stage with mixed four molecules and microspores (2) Stage with microspores (3) Stage with two nuclei of immature pollen In (1) and (2), male gametes for 20 anthers The offspring were collected. In (3), male gametes for 15 anthers were collected. In addition, after recovery, a part of each male gamete was separated and stained with DAPI to observe the number of nuclei in the male gamete and confirmed to be in each stage.
次に、このようにして調製した各雄性配偶子を、下記組成の雄性配偶子初期培地を１．３５μＬ／ｍｍ３となるように加えた親水性ＰＴＦＥ支持体（ＭＥＲＣＫ社製、製品名：Ｏｍｎｉｐｏｒｅ）上に散布し、マクロキャリアからの距離が３ｃｍの位置に配置した。
雄性配偶子初期培地の組成：０．０１６ｍＭ ホウ酸、１ｍＭ 硝酸カルシウム、１ｍＭ 硫酸マグネシウム、１０ｍＭ 硝酸カリウム、１ｗ／ｖ％ ペプトン、３ｍＭ グルタミン、１ｍＭ ウリジン、０．５ｍＭ シチジン、０．５Ｍ スクロース（２０ｍＭ リン酸緩衝液（ｐＨ７．０）にて調整）（Ｊａｒｏｓｌａｖ Ｔｕｐｙ ａｎｄ Ｌｕｄｍｉｌａ Ｒｉｈｏｖａ、Ｓｅｘｕａｌ Ｐｌａｎｔ Ｒｅｐｒｏｄｕｃｔｉｏｎ、１９９１年、４巻、２８４～２８７ページ．参照のほど）。
Next, each male gamete prepared in this manner was added with a male gamete initial medium having the following composition so as to be 1.35 μL / mm 3 , a hydrophilic PTFE support (manufactured by MERCK, product name: Omnipore). ) And placed at a
Composition of male mating initial medium: 0.016 mM borate, 1 mM calcium nitrate, 1 mM magnesium sulfate, 10 mM potassium nitrate, 1 w / v% peptone, 3 mM glutamine, 1 mM uridine, 0.5 mM cytidine, 0.5 M sulphate (20 mM phosphoric acid) Adjusted with buffer (pH 7.0)) (Jaroslav Tapy and Ludmila Rihova, Sexual Plant Reproduction, 1991, Vol. 4, pp. 284-287.).
そして、１，１００ｐｓｉ用のラプチャーディスクを用い、ヘリウムガス圧１，１００ｐｓｉ，減圧－２５～２７ｉｎＨｇで１回ずつ撃ち込みを行なった。次いで、親水性ＰＴＦＥ支持体上の花粉を雄性配偶子初期培地にて洗い流すように回収し、当該培地を用い、遮光して室温にて約２０時間培養した。その後、蛍光顕微鏡下で各雄性配偶子におけるＧＦＰ蛍光の発現を観察した。 Then, using a rupture disc for 1,100 psi, the helium gas pressure was 1,100 psi, and the pressure was reduced to -25 to 27 inHg. Then, the pollen on the hydrophilic PTFE support was collected so as to be washed away with a male gamete initial medium, and the medium was used to incubate at room temperature for about 20 hours in the dark. Then, the expression of GFP fluorescence in each male gamete was observed under a fluorescence microscope.
その結果、図４Ａ～４Ｃに示すとおり、全てのステージにて、雄性配偶子におけるＧＦＰ蛍光が検出され、本発明によれば、成熟、未成熟問わず、花粉に物質を導入できることを確認することができた。 As a result, as shown in FIGS. 4A to 4C, GFP fluorescence in male gametes was detected at all stages, and according to the present invention, it was confirmed that the substance could be introduced into pollen regardless of whether it was mature or immature. Was done.
（実施例５）
本発明の方法が、ヌクレオチドのみを対象とするものではないことを確認すべく、花粉へのその他の物質（ＲＮＡタンパク質複合体）の導入を、以下に示す方法にて試みた。
(Example 5)
In order to confirm that the method of the present invention is not intended only for nucleotides, an attempt was made to introduce another substance (RNA protein complex) into pollen by the method shown below.
＜パーティクルボンバードメント法＞
ＲＮＡタンパク質複合体（以下「ＲＮＰ」とも称する）を花粉（成熟花粉）に導入させるために、以下の操作により、ＲＮＰを装填したパーティクルボンバードメント用マクロキャリアを調製した。
<Particle bombardment method>
In order to introduce the RNA protein complex (hereinafter, also referred to as “RNP”) into pollen (mature pollen), a macro carrier for particle bombardment loaded with RNP was prepared by the following operation.
市販品であるＣａｓ９タンパク質 ３５００ｎｇ、ｔｒａｃｒＲＮＡ ４６７ｎｇ及びｃｒＲＮＡ ２８０ｎｇを、３０μｌの溶液（２０ｍＭ Ｔｒｉｓ－ＨＣｌ，２００ｍＭ ＫＣｌ，２％ スクロース）において混合し、ＲＮＰを調製した。なお、ＲＮＰの導入が検出できるよう、前記ｔｒａｃｒＲＮＡは、カルボキシフルオレセイン（ＦＡＭ：緑色蛍光）で蛍光標識化された市販品（株式会社ファスマック製）を使用した。 Commercially available Cas9 protein 3500 ng, tracrRNA 467 ng and crRNA 280 ng were mixed in a 30 μl solution (20 mM Tris-HCl, 200 mM KCl, 2% sucrose) to prepare RNP. As the tracrRNA, a commercially available product (manufactured by Fasmac Co., Ltd.) fluorescently labeled with carboxyfluorescein (FAM: green fluorescence) was used so that the introduction of RNP could be detected.
次いで、限外ろ過法により、前記ＲＮＰ溶液を、２０ｍＭ Ｔｒｉｓ及び２％スクロースからなる溶液に置換し、その後２０ｍＭ Ｔｒｉｓへの置換を行なった。一方、０．６μｍ径の金粒子（ＢＩＯ－ＲＡＤ社製）を１００％エタノールで洗浄し、滅菌水で懸濁して３０ｍｇ／ｍｌ金溶液を調製した。そして、前記緩衝液交換後のＲＮＰ溶液２０μｌへ、金溶液を１回の撃ち込みにつき１０μｌを加え、パーティクルボンバードメント用マクロキャリア（ＢｉｏＲａｄ社製 ＰＤＳ－１０００／Ｈｅ 用マクロキャリア）１枚に、３０μｌ分を装填した。装填したマクロキャリアを約１００ｈＰａの減圧下にて（１気圧の約１／１０）、２５℃、約２０分で脱気乾燥してＲＮＰを装填したパーティクルボンバードメント用マクロキャリアを作製した。 Then, the RNP solution was replaced with a solution consisting of 20 mM Tris and 2% sucrose by an ultrafiltration method, and then replaced with 20 mM Tris. On the other hand, gold particles having a diameter of 0.6 μm (manufactured by BIO-RAD) were washed with 100% ethanol and suspended in sterile water to prepare a 30 mg / ml gold solution. Then, add 10 μl of the gold solution to 20 μl of the RNP solution after exchanging the buffer solution for each shot, and add 30 μl to one particle bombardment macro carrier (PDS-1000 / He macro carrier manufactured by BioRad). Was loaded. The loaded macro carrier was degassed and dried under a reduced pressure of about 100 hPa (about 1/10 of 1 atm) at 25 ° C. for about 20 minutes to prepare a macro carrier for particle bombardment loaded with RNP.
次に、導入装置（ＰＤＳ－１０００／Ｈｅ、ＢＩＯ－ＲＡＤ社、＃１６５－２２５７ Ｊ１）本体の最上段、すなわちラプチャーディスクからの距離が３ｃｍの位置となるよう、前記マクロキャリアを配置した。生育しているベンサミアナタバコの花から成熟花粉（葯３個分、開葯後の葯から採取した雄原細胞を持つ花粉）を採取し、前記雄性配偶子初期培地を１．３５μＬ／ｍｍ３となるように加えた親水性ＰＴＦＥ支持体（ＭＥＲＣＫ社製、製品名：Ｏｍｎｉｐｏｒｅ）上に散布し、マクロキャリアからの距離が３ｃｍの位置に配置した。 Next, the macro carrier was arranged so that the distance from the uppermost stage of the main body of the introduction device (PDS-1000 / He, BIO-RAD, # 165-2257 J1), that is, the rupture disk was 3 cm. Mature pollen (three anthers, pollen with male progenitor cells collected from the anthers after opening) was collected from the growing Bensamiana tobacco flowers, and the initial gamete medium for male gametes was 1.35 μL / mm. It was sprayed on a hydrophilic PTFE support (manufactured by MERCK, product name: Stamen) added so as to be No. 3 , and placed at a position at a distance of 3 cm from the macro carrier.
そして、１，１００ｐｓｉ用のラプチャーディスクを用い、ヘリウムガス圧１，１００ｐｓｉ，減圧－２５～２７ｉｎＨｇで１回ずつ撃ち込みを行なった。次いで、蛍光顕微鏡下で、前記支持体上の花粉におけるＦＡＭ蛍光の発現を観察した。 Then, using a rupture disc for 1,100 psi, the helium gas pressure was 1,100 psi, and the pressure was reduced to -25 to 27 inHg. Then, under a fluorescence microscope, the expression of FAM fluorescence in the pollen on the support was observed.
その結果、図５に示すとおり、花粉においてＦＡＭ蛍光が検出され、本発明によれば、物質の種類を問わず、花粉に導入できることを確認することができた。 As a result, as shown in FIG. 5, FAM fluorescence was detected in pollen, and it was confirmed that according to the present invention, it can be introduced into pollen regardless of the type of substance.
以上説明したように、本発明によれば、生存率及び導入効率高く、花粉に物質を導入することが可能となる。 As described above, according to the present invention, it is possible to introduce a substance into pollen with high survival rate and introduction efficiency.
例えば、体細胞を対象とする遺伝子組換え等による植物の作出においては、長期間の培養を要する。これに対し、本発明においては、花粉を対象とするため、遺伝子系等の物質を導入した花粉を受粉させることによって、より短期間かつ簡便に後代を得ることができる。したがって、生存率及び導入効率高く、花粉に遺伝子等の物質を導入することが可能となり、さらに、受粉法により後代を簡便かつ短期間に作出できることは、農業分野等において大きく貢献するものである。 For example, long-term culture is required for the production of plants by gene recombination or the like for somatic cells. On the other hand, in the present invention, since pollen is targeted, a progeny can be easily obtained in a shorter period of time by pollinating pollen into which a substance such as a gene system has been introduced. Therefore, it is possible to introduce substances such as genes into pollen with high survival rate and introduction efficiency, and it is possible to easily produce progeny in a short period of time by pollination method, which greatly contributes to the agricultural field and the like.
１…パーティクルボンバードメント装置、２…ガス加速管、３…ラプチャーディスク、３ａ…破裂後のラプチャーディスク、３ｂ…破裂後のラプチャーディスク、４…マクロキャリア、５…物質で被覆した微粒子、６…ストッピングスクリーン、７…花粉、８…支持体。 1 ... Particle bombardment device, 2 ... Gas accelerating tube, 3 ... Rupture disk, 3a ... Rupture disk after rupture, 3b ... Rupture disk after rupture, 4 ... Macrocarrier, 5 ... Fine particles coated with substance, 6 ... Topping screen, 7 ... pollen, 8 ... support.
Claims (3)
パーティクルボンバードメント法により、物質で被覆した微粒子を支持体に載せた花粉に撃ち込む工程を含み、かつ
前記支持体が、親水性ポリテトラフルオロエチレン及び親水性混合セルロースエステルのうちの少なくとも１の高分子を含む支持体である、方法。 It ’s a way to introduce substances into pollen.
By the particle bombardment method, a step of shooting fine particles coated with a substance into pollen placed on a support is included, and the support is a polymer of at least one of hydrophilic polytetrafluoroethylene and hydrophilic mixed cellulose ester. A support, including a method.
The method according to claim 1 or 2, wherein the fine particles are metal fine particles.
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