JP5760221B2 - How to get apple pollen - Google Patents
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- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
Description
本願発明は、発芽能力を有するリンゴ花粉を、短期間で取得する方法に関するものである。 The present invention relates to a method for acquiring apple pollen having a germination ability in a short period of time.
植物は固着生物であるが故、周囲からの多様な情報を取り入れ最適な環境で発生と生長を行う必要がある。ことに花芽の形成は茎頂分裂組織における栄養生長から生殖生長への切り換えという劇的な変化を意味する発生プログラム過程の一つであり、有性生殖による繁殖を成功させる上で、この変化を決定するタイミングは特に重要である。例えばリンゴでは、種子が発芽してから６〜１２年という非常に長い栄養成長期を経て、初めて開花に至る。これは遺伝的に決められているもので、その詳細なメカニズムは不明であるが、リンゴの品種改良などにとっては非常に大きな障害となっている。 Since plants are fixed organisms, it is necessary to take in various information from the surroundings and to generate and grow them in an optimal environment. In particular, flower bud formation is one of the developmental program processes that means a dramatic change from vegetative growth to reproductive growth in the shoot apical meristem. The timing of determination is particularly important. For example, in apples, flowering occurs only after a very long vegetative growth period of 6 to 12 years after seeds germinate. This is genetically determined and its detailed mechanism is unknown, but it is a huge obstacle for improving apple varieties.
花芽形成開始のタイミングは栄養状態や概日リズム、また植物の生育ステージなどといった内的要因と、温度や光周期などの環境による外的要因に制御されていることが明らかにされている(非特許文献1)。近年では、モデル植物で、長日植物のArabidopsis thaliana(以下シロイヌナズナ)を用いた精力的な研究により、光周期(photoperiodic)、春化(vernalization)、ジベレリン(GA)、そして自律的(autonomous)花芽形成促進の4つの経路が花芽形成を決定すると提唱されている(非特許文献2-4)。これらの経路の各シグナルは必要
に応じて補い合うように働いているため、どれか一つの機能が失われたとしても花芽形成が完全に阻害されることはない。また、花芽形成のシグナルは、FLOWERING LOCUS T(FT)遺伝子とSUPPRESSOR OF OVEREXPRESSION OF CO1(SOC1)遺伝子に統合され、花の形態形成を誘導するAPETALA1(AP1)遺伝子とLEAFY(LFY)遺伝子の発現を促進し、開花を促す(非特許文献5-7)。経路統合遺伝子であるFT遺伝子は光周期花芽促進経路により強く発現される遺伝子であり、この遺伝子の転写制御は花芽形成を調節する上で最も重要なステップである。
It has been clarified that the timing of flower bud formation is controlled by internal factors such as nutritional status, circadian rhythm, and plant growth stage, and by external factors such as temperature and photoperiod (non- Patent Document 1). In recent years, a vigorous study using the long-day plant Arabidopsis thaliana (Arabidopsis thaliana) as a model plant has led to photoperiodic, vernalization, gibberellin (GA), and autonomous flower buds. It has been proposed that four pathways for promoting formation determine flower bud formation (Non-patent Documents 2-4). Since each signal of these pathways works to complement as needed, even if one of the functions is lost, flower bud formation is not completely inhibited. In addition, the signal of flower bud formation is integrated into the FLOWERING LOCUS T (FT) gene and the SUPPRESSOR OF OVEREXPRESSION OF CO1 (SOC1) gene, and the expression of the APETALA1 (AP1) and LEAFY (LFY) genes that induce flower morphogenesis Promotes and promotes flowering (Non-Patent Documents 5-7). The FT gene, which is a pathway integration gene, is a gene that is strongly expressed by the photoperiodic flower bud promotion pathway, and transcriptional control of this gene is the most important step in regulating flower bud formation.
FT遺伝子は1991年に花芽形成遅延変異体の解析から明らかにされた(非特許文献8)。概日時計と光受容体の相互作用によって日長の変化を受容する場所は葉であることが知られており、葉の師部組織でCONSTANS（CO）遺伝子の発現が誘導され(非特許文献6、9)、さらにCO遺伝子の発現がFT遺伝子の発現を促進する(非特許文献10-14)。このようにして葉で発現したFT遺伝子はFTタンパク質の形で茎頂分裂組織に移行することが明らかとなっている (非特許文献15)。また、茎頂で特異的に発現するFD遺伝子はbZIP型転写因子をコードしており、FDタンパク質は核に局在することが確認されている(非特許文献16、17)。FDタンパク質の制御標的は花芽分裂調節遺伝子であるAP1遺伝子であり、FTタンパク質がこのFDタンパク質と結合することでAP1遺伝子の転写活性を促進し、花芽形成を促進する(非特許文献16、18)。 The FT gene was revealed in 1991 from the analysis of flower bud formation delay mutants (Non-patent Document 8). It is known that the place where the change of day length is received by the interaction of circadian clock and photoreceptors is the leaf, and the expression of CONSTANS (CO) gene is induced in the phloem tissue of the leaf (non-patent literature) 6, 9) Furthermore, the expression of the CO gene promotes the expression of the FT gene (Non-patent Documents 10-14). Thus, it has been clarified that the FT gene expressed in the leaves is transferred to the shoot apical meristem in the form of FT protein (Non-patent Document 15). Moreover, the FD gene specifically expressed at the shoot apex encodes a bZIP type transcription factor, and it has been confirmed that the FD protein is localized in the nucleus (Non-patent Documents 16 and 17). The control target of the FD protein is the AP1 gene, which is a flower bud division regulatory gene, and the FT protein binds to this FD protein to promote the transcriptional activity of the AP1 gene and promote flower bud formation (Non-patent Documents 16 and 18). .
一方、FT遺伝子と高い相同性を示すシロイヌナズナのTERMINAL FLOWER 1(TFL1)遺伝子は、FT遺伝子とは逆に花芽形成を抑制することが明らかにされている(非特許文献19、20)。TFL1遺伝子がその高い相同性のためFT遺伝子と拮抗し、FDタンパク質と結合することでAP1遺伝子の転写活性を阻害するためである(非特許文献21)。このTFL1遺伝子の発現抑制が花芽形成を促進することが、TFL1遺伝子を不活化した変異体の形質転換体を作出することにより実証された(非特許文献22)。また最近ではリンゴのTFL1(MdTFL1)遺伝子のアンチセンス鎖を導入した変異体が、実際にリンゴで花芽形成を促進した例が報告され、これはTFL1遺伝子のサイレンシングによるものと推定された(非特許文献23)。RNAサイレンシングによる遺伝子の発現調節は、今後この花芽形成調節機構を解明する遺伝学的アプローチとして有効な手段となるであろう。 On the other hand, it has been clarified that the Arabidopsis TERMINAL FLOWER 1 (TFL1) gene showing high homology with the FT gene suppresses flower bud formation, contrary to the FT gene (Non-patent Documents 19 and 20). This is because the TFL1 gene antagonizes the FT gene because of its high homology, and inhibits the transcriptional activity of the AP1 gene by binding to the FD protein (Non-patent Document 21). It has been demonstrated that the suppression of TFL1 gene expression promotes flower bud formation by producing a mutant transformant in which the TFL1 gene is inactivated (Non-patent Document 22). Recently, an example in which an antisense strand of the apple TFL1 (MdTFL1) gene introduced an antisense strand actually promoted flower bud formation in apples was presumed to be due to TFL1 gene silencing (non- Patent Document 23). Regulation of gene expression by RNA silencing will be an effective genetic approach to elucidate this mechanism of flower bud formation.
リンゴ小球形潜在ウイルス（Apple latent spherical virus：ALSV）は2分節の1本鎖RNAゲノム(RNA1とRNA2)と3種類の外被タンパク質（Vp25、Vp20、Vp24）から構成される径25nmのウイルスであり、リンゴ以外に、5種のナス科植物[Nicotiana tabacum cv. Xanthi nc(以下タバコ)、Nicotiana glutinosa(以下グルチノーサ)、Nicotiana occidentalis(以下オキシデンタリス)、ベンサミアナ、ペチュニア]やシロイヌナズナなどに潜在感染することが明らかにされている(非特許文献24)。ALSVは実験植物であるChenopodium quinoa(以下キノア)では全身感染して葉脈透過や退緑斑紋の症状を（非特許文献25、26）、ダイズでは感染初期に退緑斑紋症状を引き起こす。これまでにALSV-RNA2がコードする細胞間移行タンパク質（MP）とVp25の間にプロテアーゼ切断サイトを反復し、外来遺伝子導入サイトを付加した感染性cDNAクローンが作出され（非特許文献27-30）、さらに、これを利用した感染植物における外来遺伝子の発現(特許文献2、非特許文献27、31、32)が報告されている。ALSVはリンゴをはじめとしてほとんどの宿主で潜在感染するというウイルスベクターとして非常に有利な特徴を持っており、様々な有用遺伝子の導入と発現、さらにはVIGSを利用したポストゲノム解析や、原宿主によるリンゴの育種への応用など多くの可能性が期待されている。 Apple latent spherical virus (ALSV) is a 25-nm diameter virus composed of a two-segment single-stranded RNA genome (RNA1 and RNA2) and three coat proteins (Vp25, Vp20, and Vp24). In addition to apples, there are five types of solanaceous plants (Nicotiana tabacum cv. Xanthi nc (tobacco), Nicotiana glutinosa) (Non-patent Document 24). ALSV is a systemic infection in the experimental plant Chenopodium quinoa (hereinafter referred to as quinoa) and causes leaf vein penetration and erythema mottle (Non-Patent Documents 25 and 26). So far, an infectious cDNA clone has been created in which a protease cleavage site is repeated between the cell-to-cell translocation protein (MP) encoded by ALSV-RNA2 and Vp25, and a foreign gene introduction site is added (Non-patent Documents 27-30) Furthermore, the expression of foreign genes in infected plants using this (Patent Document 2, Non-Patent Documents 27, 31, and 32) has been reported. ALSV has a very advantageous feature as a viral vector that latently infects most hosts, including apples, and introduces and expresses various useful genes, as well as post-genome analysis using VIGS and depending on the original host. Many possibilities such as application to breeding apples are expected.
我が国の主要果樹であるリンゴやナシ等のバラ科果樹は播種から開花まで通常6〜１２年を要し、これがリンゴやナシの育種（品種改良）を困難にしている最大の原因の一つである。 The main fruit trees of our country, apples and pears such as roses, usually take 6 to 12 years from sowing to flowering, and this is one of the biggest causes of difficulty in breeding apples and pears. is there.
近年リンゴにおいても、花芽形成に関係する遺伝子機構が解明されつつあり、前記のとおり、花芽形成を抑制するリンゴTFL1遺伝子の発現抑制がリンゴの花芽形成を促進することが報告されている。すなわち、リンゴ（品種：王林）について、リーフディスク法を用いてTFL1遺伝子のアンチセンス鎖を導入し、組織培養とシュート再生を行った後に得られた形質転換リンゴのシュートを矮性台木に接ぎ木し、その後、温室に搬入して育成した形質転換リンゴは、同条件下で育成した非形質転換リンゴが温室搬入後に開花まで約6年（69ヶ月）を要したのに対して、温室搬入後に8〜25ヶ月で開花した（非特許文献23）。しかしながら、リンゴの形質転換には多大な労力、並びに組織培養とシュート再生に長い期間を要するうえに、形質転換効率も低く（非特許文献23におけるリンゴの形質転換効率は0.15％）、さらに、形質転換が可能なリンゴ品種は限られているのが現状である。また、形質転換リンゴの導入遺伝子は次世代にも受け継がれるため、形質転換植物に対する規制が厳しい現在の情勢では、早期開花する形質転換リンゴが得られても、その形質転換リンゴから得られた次世代個体をそのまま育種素材として使用することは不可能である。 In recent years, gene mechanisms related to flower bud formation have been elucidated in apples, and as described above, it has been reported that suppression of apple TFL1 gene expression that suppresses flower bud formation promotes apple flower bud formation. In other words, for apples (variety: Wang Lin), the TFL1 gene antisense strand was introduced using the leaf disc method, and the shoots of transformed apples obtained after tissue culture and shoot regeneration were grafted onto dwarf rootstocks. After that, the transformed apples brought up and grown in the greenhouse took about 6 years (69 months) to flower after the untransformed apples grown under the same conditions. It bloomed in 8-25 months (Non-patent Document 23). However, the transformation of apples requires a great deal of labor, a long period of time for tissue culture and shoot regeneration, and the transformation efficiency is low (the transformation efficiency of apples in Non-Patent Document 23 is 0.15%). The current situation is that apple varieties that can be converted are limited. In addition, since the transgenes of transformed apples are inherited by the next generation, even if transformed apples that flower early are obtained in the current situation where regulations on transformed plants are strict, It is impossible to use generational individuals as breeding materials.
一方、ウイルスベクターによる植物への外来遺伝子導入は、外来遺伝子を組み込んだウイルスが植物に感染・増殖することで成立するため、形質転換法と比較すると簡便性と迅速性に富んでいる。有効に機能するウイルスベクターの条件として、感染植物に激しい病徴を引き起こさないことや、感染植物において安定して増殖することなどが挙げられるが、ALSVベクターはこれらの条件を満たすウイルスベクターである。すなわち、ALSVベクターはリンゴに病気を引き起こすことなく無病徴感染し、安定して全身感染を維持する。ALSVベクターを用いた外来遺伝子の導入技術（例えば、特許文献２）が知られているが、このALSVベクター技術を用いたバラ科果樹の開花促進は全く知られていない。また、一般的に果樹類への安定したウイルス接種は困難であり、ALSVについてもリンゴをはじめとするバラ科果樹への効率的接種法はこれまでに確立されておらず、このことがバラ科果樹におけるALSVベクターを用いた外来遺伝子導入技術の利用への大きな障害になっていた。しかしながら、前記のとおり、ALSVベクターはリンゴをはじめとするバラ科果樹のウイルスベクターとして優れた特性を有している。また、ALSVは種子伝染するがその種子伝染率は低率であることから、ALSVベクター技術を用いて開花促進がなされたリンゴの次世代からはウイルスフリーの個体を選抜することが可能であり、その選抜したウイルスフリー個体は、遺伝子導入がなされていないリンゴとなんら変わることがないことから、そのまま育種素材として使用することが可能である。したがって、ALSVベクター技術によるリンゴをはじめとするバラ科果樹の開花促進法が確立できれば、即役立つ実用技術となると期待できる。 On the other hand, introduction of a foreign gene into a plant by a viral vector is achieved by infecting and multiplying the plant with a virus incorporating the foreign gene, and is therefore simpler and quicker than the transformation method. Conditions for a virus vector that functions effectively include not causing severe symptoms in the infected plant and stable growth in the infected plant. The ALSV vector is a virus vector that satisfies these conditions. That is, the ALSV vector can be transmitted asymptomatically without causing illness in apples and stably maintain systemic infection. A technique for introducing a foreign gene using an ALSV vector (for example, Patent Document 2) is known, but no promotion of flowering of a Rosaceae fruit tree using this ALSV vector technique is known at all. In addition, stable virus inoculation of fruit trees is generally difficult, and an efficient method of inoculating rose fruit fruits including apples has not been established for ALSV. This was a major obstacle to the use of foreign gene transfer technology using ALSV vectors in fruit trees. However, as described above, the ALSV vector has excellent characteristics as a viral vector for rose fruit trees including apples. In addition, since ALSV is seed-transmitted but its seed transmission rate is low, it is possible to select virus-free individuals from the next generation of apples that have been promoted to flower using ALSV vector technology, The selected virus-free individuals can be used as breeding materials as they are because they are not different from apples into which no gene has been introduced. Therefore, if a method for promoting flowering of fruit trees such as apples using ALSV vector technology can be established, it can be expected to be a practical technology that is immediately useful.
本願発明は、以上のとおりの事情に鑑みてなされたものであり、リンゴの開花を促進することによって、短期間でリンゴの花粉を取得するための新しい手段を提供することを課題としている。 This invention is made | formed in view of the above situations, and makes it a subject to provide the new means for acquiring an apple pollen in a short period of time by promoting the flowering of an apple.
本願は、前記の課題を解決するための発明として、シロイヌナズナFT遺伝子を発現する組換えリンゴ小球形潜在ウイルス（FT-ALSV）に感染した増殖宿主から濃縮したウイルスRNAを、発根直後のリンゴ実生の子葉にパーティクルガン法を用いて接種する工程を含み、播種から1.5ヵ月〜3ヵ月で開花させ、この花から発芽能力を有する花粉を採取することを特徴とするリンゴの花粉取得方法を提供する。 In the present application, as an invention for solving the above-mentioned problems, viral RNA concentrated from a propagation host infected with a recombinant apple small spherical latent virus (FT-ALSV) expressing an Arabidopsis FT gene is used as an apple seedling immediately after rooting. Providing a method for obtaining apple pollen, comprising the step of inoculating a cotyledon of a rice plant using a particle gun method, wherein the seedlings are allowed to bloom 1.5 to 3 months after sowing, and pollen having germination ability is collected from the flower .
本願発明によれば、通常は６〜12年を要するリンゴの開花を、1.5〜3ヶ月と大幅に短縮することを可能とする。これによって、リンゴの品種改良を効率よく行うことが可能となる。 According to the present invention, the flowering of apples, which normally requires 6 to 12 years, can be significantly shortened to 1.5 to 3 months. This makes it possible to efficiently improve the variety of apples.
また、前記いずれかの方法で開花促進されたバラ科果樹の次世代個体から選別されたウイルスフリーの個体は、遺伝子導入がなされていないリンゴ個体となんら変わることがないことから、この選別個体やその種子はそのまま育種素材として使用することができる。 In addition, since virus-free individuals selected from the next-generation individuals of the Rosaceae fruit tree whose flowering has been promoted by any of the above methods will not change at all from apple individuals that have not been introduced with the gene, The seed can be used as it is as a breeding material.
シロイヌナズナFT遺伝子は、公知の配列情報（GenBank/AB027504）に基づいて、シロイヌナズナのトータルRNAを鋳型とするRT-PCRや、シロイヌナズナcDNAライブラリーのプラークハイブリダイゼーション法等の公知の方法により取得することができる。具体的には、後記の実施例に記載の手順で容易に取得することができる。 Based on known sequence information (GenBank / AB027504), the Arabidopsis FT gene can be obtained by a known method such as RT-PCR using Arabidopsis thaliana total RNA as a template or a plaque hybridization method of an Arabidopsis cDNA library. it can. Specifically, it can be easily obtained by the procedure described in the examples described later.
FT遺伝子を発現する組換えALSVベクター（FT-ALSV）の作製は、基本的には特許文献２に開示された方法に従って行うことができる。すなわち、ALSV RNA2の感染性cDNAクローンであるpEALSR2L5R5の外来遺伝子導入サイトに、FT遺伝子 cDNAを挿入することによってpEALSR2L5R5FTを構築し、ALSV RNA1の感染性cDNAクローンであるpEALSR1とともに増殖宿主に接種しウイルス化を行うことでFT-ALSVを得ることができる。 Production of a recombinant ALSV vector (FT-ALSV) that expresses the FT gene can be basically performed according to the method disclosed in Patent Document 2. In other words, pEALSR2L5R5FT was constructed by inserting the FT gene cDNA into the foreign gene transfer site of pEALSR2L5R5, an infectious cDNA clone of ALSV RNA2, and inoculated into a growth host together with pEALSR1, an infectious cDNA clone of ALSV RNA1, and made viral. FT-ALSV can be obtained.
このようにして得たFT-ALSV感染キノア用からFT-ALSVを濃縮し、濃縮試料から抽出したRNAをパーティクルガン法でバラ科果樹実生の子葉に接種することで、ほぼ100%の効率でFT-ALSV感染実生苗を作出でき、リンゴ等のバラ科果樹の開花を大幅に早めることが可能となる。本願発明におけるFT-ALSVのパーティクルガン接種は以下の手順で行うことができる。
工程(1)：FT-ALSVを増殖宿主（例えば、キノア）に接種する。
工程(2)：増殖宿主の感染葉からFT- ALSVを抽出し、遠心分離等の処理により濃縮する。
工程(3)：この濃縮したFT- ALSVからRNAを単離する。
工程(4)：発根直後のバラ科果樹植物実生の子葉にパーティクルガン法を用いて前記RNAを接種する。
By concentrating FT-ALSV from the FT-ALSV-infected quinoa obtained in this way and inoculating the RNA extracted from the concentrated sample into the cotyledons of Rosaceae fruit tree seedlings by the particle gun method, the efficiency of FT is almost 100%. -AlSV-infected seedlings can be produced, and the flowering of apples and other rose family fruit trees can be greatly accelerated. The particle gun inoculation of FT-ALSV in the present invention can be performed by the following procedure.
Step (1): Inoculate FT-ALSV into a growth host (eg, quinoa).
Step (2): FT-ALSV is extracted from the infected leaves of the proliferating host and concentrated by a treatment such as centrifugation.
Step (3): RNA is isolated from the concentrated FT-ALSV.
Step (4): Inoculate the cotyledons of a rose family fruit tree seedling immediately after rooting with the RNA using a particle gun method.
これらの具体的操作は、後記実施例に詳細に記載されており、実施例の記載に従って実施することができるが、特にこの方法は、前記工程(3)においてFT-ALSVから単離したRNAを接種すること、並びに前記工程（4）においてバラ科果樹実生の子葉に接種することを特徴の一つとしている。 These specific operations are described in detail in Examples described later, and can be carried out according to the description in the Examples. In particular, this method involves the isolation of RNA isolated from FT-ALSV in the above step (3). One of the features is inoculation, and inoculation of cotyledons of rose fruit seedlings in the step (4).
また、工程(4)における「発根直後のバラ科果樹植物実生の子葉」は以下の様に調製する。先ず、休眠終了後の発根間もない実生苗の種子の種皮をメスで除去し、子葉を露出させる。そして、RNAを塗布したマイクロキャリアをパーティクルガンを用いて子葉に接種する。このようにして接種したFT-ALSVは、100％の効率でバラ科果樹実生に感染する。 In addition, the “cotyledon of a rose family fruit tree seedling immediately after rooting” in step (4) is prepared as follows. First, seed coats of seedlings that have just been rooted after the end of dormancy are removed with a scalpel to expose the cotyledons. Then, the microcarriers coated with RNA are inoculated into cotyledons using a particle gun. FT-ALSV inoculated in this way infects rose tree seedlings with 100% efficiency.
RNAを接種した種子は、遮光して湿度を保った状態で2〜3日静置したのち、徐々に外気に馴化させ、その後培養土に移植して、通常の生育温度（約25℃）で育成する。 Seeds inoculated with RNA are allowed to stand for 2 to 3 days in a light-shielded and humidified condition, then gradually acclimatize to the outside air, and then transplanted to culture soil at a normal growth temperature (about 25 ° C). Cultivate.
このようにして育成したバラ科果樹は、後記の実施例に示したリンゴの場合は約40％の個体が1.5〜3ヶ月で開花した。 In the case of the apples shown in the examples described later, about 40% of the rose fruit trees grown in this manner flowered in 1.5 to 3 months.
以下、実施例を示して本願発明をさらに詳細かつ具体的に説明するが、本願発明は以下の例によって限定されるものではない。 EXAMPLES Hereinafter, although an Example is shown and this invention is demonstrated further in detail and concretely, this invention is not limited by the following examples.
1．材料および方法
(1) FT-ALSVの感染性cDNAクローンの構築
シロイヌナズナFT遺伝子(864bp,accession number:AB027504)のFTタンパク質発現領域である525bp(塩基番号70〜594番)を次のように増幅した。DNA増幅の際、鋳型にはpBlue script II SK(+)のXbaＩ/SacＩサイトにFT mRNAの塩基番号29〜709番までの配列を組み込んだプラスミド(pBSAtFT-19)を用い、またプラス鎖プライマーには10μM FT-Xho(+)[5’- CCGCTCGAGATGTCTATAAATATAAGAGA-3’]（配列番号１）を、マイナス鎖プライマーには10μM FT-Sma(-)[5’-TCCCCCGGGAAGTCTTCTTCCTCCGCAGC-3’]（配列番号２）を用いた。鋳型DNA溶液(10ng/μl)を1μl、プラス鎖プライマーとマイナス鎖プライマーをそれぞれ2μl、2.5mM dNTP mixture(TaKaRa)を1.6μl、10×Ex Taq Buffer(TaKaRa)を2μl、滅菌水を11.2μl、TaKaRa Ex Taqを0.2μl混合し、GeneAmp PCR System2400(Perkin Elmer)を使用して94℃で5分間処理した後、[94℃、30秒→55℃、30秒→72℃、60秒]の反応を35サイクル行い、続いて72℃で7分間処理した後、最後に4℃で5分間処理してPCRを終了した。得られたPCR産物1μlにLoading Buffer[0.25％ブロモフェノールブルー,1mM EDTA(pH8.0),40％スクロース]1μlを加え、Agarose S(ニッポンジーン)0.15g、TAE[40mM Tris,20mM 酢酸,1mM EDTA(pH8.0)]15ml、エチジウムブロマイド0.6μlで調製した1％アガロースゲルのウェルにアプライして電気泳動し、増幅したDNAが期待されたFT遺伝子のサイズであることを確認した。
1． Materials and methods
(1) Construction of FT-ALSV infectious cDNA clone 525 bp (base numbers 70 to 594), which is the FT protein expression region of Arabidopsis FT gene (864 bp, accession number: AB027504), was amplified as follows. During DNA amplification, a plasmid (pBSAtFT-19) in which the sequences from base numbers 29 to 709 of FT mRNA were incorporated into the XbaI / SacI site of pBluescript II SK (+) was used as the template, and the positive strand primer Is 10 μM FT-Xho (+) [5′-CCGCTCGAGATGTCTATAAATATAAGAGA-3 ′] (SEQ ID NO: 1), and 10 μM FT-Sma (−) [5′-TCCCCCGGGAAGTCTTCTTCCTCCGCAGC-3 ′] (SEQ ID NO: 2) Was used. 1 μl of template DNA solution (10 ng / μl), 2 μl each of positive and negative strand primers, 1.6 μl of 2.5 mM dNTP mixture (TaKaRa), 2 μl of 10 × Ex Taq Buffer (TaKaRa), 11.2 μl of sterile water, After mixing with 0.2 μl of TaKaRa Ex Taq and treating with GeneAmp PCR System2400 (Perkin Elmer) at 94 ° C for 5 minutes, [94 ° C, 30 seconds → 55 ° C, 30 seconds → 72 ° C, 60 seconds] reaction 35 cycles, followed by treatment at 72 ° C. for 7 minutes, and finally treatment at 4 ° C. for 5 minutes to complete PCR. Add 1 μl of Loading Buffer [0.25% bromophenol blue, 1 mM EDTA (pH 8.0), 40% sucrose] to 1 μl of the obtained PCR product, 0.15 g of Agarose S (Nippon Gene), TAE [40 mM Tris, 20 mM acetic acid, 1 mM EDTA (pH 8.0)] It was applied to a well of a 1% agarose gel prepared with 15 ml and 0.6 μl of ethidium bromide and electrophoresed to confirm that the amplified DNA had the expected FT gene size.
次に、増幅したFT遺伝子をXhoＩおよびSmaＩで以下のように切断した。まず、PCRで増幅したFT遺伝子の溶液を10μl、10×K Buffer(TaKaRa)を10μl、滅菌水を78μl、XhoＩ(TaKaRa)を2μl混合し、37℃で2時間静置した。この反応液に100μlの滅菌水、100μlのTE[10mM Tris-HCl(pH8.0)，1mM EDTA(pH8.0)]飽和フェノール、100μlのクロロホルムを順に加え、ボルテックスミキサーで30秒間撹拌し、14,000rpmで5分間(4℃)遠心分離した。上清200μlに等量のクロロホルムを加え、ボルテックスミキサーで30秒間撹拌した後、14,000rpmで5分間(4℃)遠心分離し、この上清200μlを別の1.5ml容チューブに移した。その上清に20μlの3M 酢酸ナトリウム(pH5.2)、600μlの99％エタノールを加え、十分に撹拌した後、-80℃で30分間静置した。14,000rpmで10分間(4℃)遠心分離して得られた沈殿に70％エタノール1mlを加えて14,000rpmで5分間(4℃)遠心分離した。上清を捨て、沈殿を減圧乾燥し、50μlの滅菌水に懸濁した。引き続きこの溶液に10×T Buffer(TaKaRa)を10μl、0.1％BSA(TaKaRa)を10μl、滅菌水を28μl、SmaＩ(TaKaRa)を2μl混合し、25℃で2時間静置した。制限酵素処理を施した反応液に100μlの滅菌水、100μlのTE飽和フェノール、100μlのクロロホルムを加え、ボルテックスミキサーで30秒間撹拌し、14,000rpmで5分間(4℃)遠心分離した。上清200μlに等量のクロロホルムを加え、ボルテックスミキサーで30秒間撹拌した後、14,000rpmで5分間(4℃)遠心分離した。遠心分離後、この上清200μlに20μlの3M 酢酸ナトリウム(pH5.2)と600μlの99％エタノールを加え、十分に撹拌した後、-80℃で30分間静置した。14,000rpmで10分間(4℃)遠心分離して得られた沈殿に70％エタノール1mlを加えて14,000rpmで5分間(4℃)遠心分離した。上清を捨て、沈殿を減圧乾燥し、20μlの滅菌水に懸濁した。XhoＩおよびSmaＩによる酵素処理は、ALSV-RNA2に外来遺伝子導入サイトを付加した感染性cDNAクローンのpEALSR2L5R5(100ng/μl)10μlに対しても上と同様に行った。 Next, the amplified FT gene was cut with XhoI and SmaI as follows. First, 10 μl of the FT gene solution amplified by PCR, 10 μl of 10 × K Buffer (TaKaRa), 78 μl of sterilized water, and 2 μl of XhoI (TaKaRa) were mixed and allowed to stand at 37 ° C. for 2 hours. 100 μl of sterilized water, 100 μl of TE [10 mM Tris-HCl (pH 8.0), 1 mM EDTA (pH 8.0)] saturated phenol and 100 μl of chloroform were added to the reaction solution in this order, and the mixture was stirred for 30 seconds with a vortex mixer. Centrifugation at rpm for 5 minutes (4 ° C). An equal volume of chloroform was added to 200 μl of the supernatant, stirred for 30 seconds with a vortex mixer, and then centrifuged at 14,000 rpm for 5 minutes (4 ° C.), and 200 μl of this supernatant was transferred to another 1.5 ml tube. 20 μl of 3M sodium acetate (pH 5.2) and 600 μl of 99% ethanol were added to the supernatant, and after sufficient stirring, the mixture was allowed to stand at −80 ° C. for 30 minutes. To the precipitate obtained by centrifugation at 14,000 rpm for 10 minutes (4 ° C.), 1 ml of 70% ethanol was added and centrifuged at 14,000 rpm for 5 minutes (4 ° C.). The supernatant was discarded and the precipitate was dried under reduced pressure and suspended in 50 μl of sterile water. Subsequently, 10 μl of 10 × T Buffer (TaKaRa), 10 μl of 0.1% BSA (TaKaRa), 28 μl of sterilized water, and 2 μl of SmaI (TaKaRa) were mixed with this solution and allowed to stand at 25 ° C. for 2 hours. 100 μl of sterilized water, 100 μl of TE saturated phenol, and 100 μl of chloroform were added to the reaction solution subjected to the restriction enzyme treatment, stirred for 30 seconds with a vortex mixer, and centrifuged at 14,000 rpm for 5 minutes (4 ° C.). An equal volume of chloroform was added to 200 μl of the supernatant, stirred for 30 seconds with a vortex mixer, and then centrifuged at 14,000 rpm for 5 minutes (4 ° C.). After centrifugation, 20 μl of 3M sodium acetate (pH 5.2) and 600 μl of 99% ethanol were added to 200 μl of the supernatant, and after sufficient stirring, the mixture was allowed to stand at −80 ° C. for 30 minutes. To the precipitate obtained by centrifugation at 14,000 rpm for 10 minutes (4 ° C.), 1 ml of 70% ethanol was added and centrifuged at 14,000 rpm for 5 minutes (4 ° C.). The supernatant was discarded and the precipitate was dried under reduced pressure and suspended in 20 μl of sterile water. Enzyme treatment with XhoI and SmaI was performed in the same manner as above on 10 μl of pEALSR2L5R5 (100 ng / μl), an infectious cDNA clone in which a foreign gene introduction site was added to ALSV-RNA2.
続いて、制限酵素処理したFT遺伝子およびpEALSR2L5R5の回収をQIA quick Gel Extraction Kit(QIAGEN)を用いて行った。FT遺伝子の溶液18μlに10×Loading Buffer 2μlを加え、1％アガロースゲルのウェルにアプライして電気泳動した後、メスを用いて目的のDNA断片をアガロースゲルから切り出し、1.5ml容チューブに入れゲルの重量を測った。ゲルの3倍容のBuffer QGを1.5ml容チューブに加え50℃に加温してゲルを完全に溶解し、溶液の色が黄色であることを確認した。ゲルと等量のイソプロパノールを加え、2ml容のチューブにカラムをセットし、これにゲルが溶解した溶液を入れ、10,000rpmで1分間(室温)遠心分離した。2ml容チューブに落ちた溶液を捨て、カラムに750μlのBuffer PEを加えて洗浄し、10,000rpmで1分間(室温)遠心分離した後、再度2ml容チューブに落ちた溶液を捨てた。10,000rpmで1分間(室温)遠心分離し、カラムを新たな1.5ml容チューブにセットし、DNA溶出のため30μlのBuffer EB[10mＭ Tris-HCl(pH8.5)]をカラムの中央に加え、1分間静置後、13,000rpmで1分間(室温)遠心分離した。 Subsequently, the restriction enzyme-treated FT gene and pEALSR2L5R5 were collected using the QIA quick Gel Extraction Kit (QIAGEN). Add 2 μl of 10 × Loading Buffer to 18 μl of the FT gene solution, apply it to a 1% agarose gel well, perform electrophoresis, cut out the DNA fragment of interest from the agarose gel using a scalpel, place it in a 1.5 ml tube, and place the gel Weighed. Buffer QG 3 times the volume of the gel was added to a 1.5 ml tube and heated to 50 ° C. to completely dissolve the gel, and the color of the solution was confirmed to be yellow. The same amount of isopropanol as the gel was added, the column was set in a 2 ml tube, and the solution in which the gel was dissolved was placed therein, followed by centrifugation at 10,000 rpm for 1 minute (room temperature). The solution dropped in the 2 ml tube was discarded, and 750 μl of Buffer PE was added to the column for washing. After centrifugation at 10,000 rpm for 1 minute (room temperature), the solution dropped in the 2 ml tube was discarded again. Centrifuge at 10,000 rpm for 1 minute (room temperature), set the column in a new 1.5 ml tube, add 30 μl Buffer EB [10 mM Tris-HCl (pH 8.5)] to the center of the column for DNA elution, After leaving still for 1 minute, it centrifuged at 13,000 rpm for 1 minute (room temperature).
ゲル回収後のFT遺伝子をインサートDNA、pEALSR2L5R5をプラスミドベクターとしてライゲーションを行った。インサートDNA溶液4μlとプラスミドベクター溶液1μlを混合し、DNA Ligation Kit Ver.2.1(TaKaRa)のＩ液を5μl加えて16℃で2時間静置した後、1.1μlのIII液を加えてライゲーション溶液とした。 Ligation was performed using the FT gene after gel recovery as insert DNA and pEALSR2L5R5 as a plasmid vector. Mix 4 μl of the insert DNA solution and 1 μl of the plasmid vector solution, add 5 μl of DNA Ligation Kit Ver.2.1 (TaKaRa) solution I and let stand at 16 ° C. for 2 hours, then add 1.1 μl of solution III and add the ligation solution. did.
形質転換はHeat Shock法で行った。-80℃で保存していた100μlのコンピテントセルを氷中でゆっくりと解凍し、そこへライゲーション溶液を5μl加え5秒間ゆっくりと混合し、氷中で30分間静置した。続いてウォーターバスを用いて42℃で45秒間加温し、終了後2分間氷中で冷却した。予め温めておいたSOC[2％tryptone,0.5％yeast extract,0.058％NaCl,0.019％KCl,10mM MgCl2,10mM MgSO4,20mM グルコース] 900μlをクリーンベンチ内で加え蓋をしてパラフィルムを巻き、振とう培養器を用いて37℃で1時間振とうした。この培養液200μlをLMAプレート[1％tryptone,0.5％yeast extract,0.058％NaCl,10mM MgSO4,1.5％agar,40mg/mlアンピシリン]に滴下し、スプレッダーで培地の表面に塗布し、シャーレの蓋を開けた状態で10分間乾かした。残った培養液800μlは14,000rpmで30秒間遠心分離し、上清600μlを捨て残った200μlの溶液で沈殿を懸濁した。この溶液のうち100μlを同様にLMAプレートに滴下してスプレッダーで培地の表面に塗布、さらに蓋を開けた状態で10分間乾かした。培地が乾燥したら各プレートはインキュベーターに入れ、37℃で12〜16時間培養した。 Transformation was performed by the Heat Shock method. 100 μl of the competent cell stored at −80 ° C. was thawed slowly in ice, and 5 μl of the ligation solution was added thereto, mixed gently for 5 seconds, and allowed to stand in ice for 30 minutes. Subsequently, the mixture was heated at 42 ° C. for 45 seconds using a water bath, and cooled in ice for 2 minutes after the completion. Pre-warmed SOC [2% tryptone, 0.5% yeast extract, 0.058% NaCl, 0.019% KCl, 10 mM MgCl2, 10 mM MgSO4, 20 mM glucose] 900 μl in a clean bench, cover, roll parafilm, shake The mixture was shaken at 37 ° C. for 1 hour using an incubator. 200 μl of this culture solution is dropped onto an LMA plate [1% tryptone, 0.5% yeast extract, 0.058% NaCl, 10 mM MgSO4, 1.5% agar, 40 mg / ml ampicillin], applied to the surface of the medium with a spreader, and a petri dish lid is applied. It was dried for 10 minutes in the open state. The remaining culture solution (800 μl) was centrifuged at 14,000 rpm for 30 seconds, the supernatant (600 μl) was discarded, and the remaining 200 μl solution was suspended. 100 μl of this solution was similarly dropped onto the LMA plate, applied to the surface of the medium with a spreader, and further dried for 10 minutes with the lid open. When the medium was dried, each plate was placed in an incubator and cultured at 37 ° C. for 12 to 16 hours.
形質転換したコロニーをスモールスケール培養するため2mlのLB培養液[1％tryptone,0.5％yeast extract,1％NaCl]を入れた試験管を16本用意し、オートクレーブ後これに10倍希釈したアンピシリン[25mg/ml]を各試験管に40μl添加した。コロニーを滅菌した爪楊枝の先端でかきとりLMAプレートに接触させることで大腸菌の一部を植菌してマスタープレートを作成し、爪楊枝はそのままLB培養液の入った試験管に入れた。マスタープレートはコロニーごとに番号をふって区別し、37℃で一晩静置培養した後、ビニールテープで密閉し、4℃で保存した。LB培養液の入った試験管にもマスタープレートと対応する番号をふり、傾けて37℃で一晩振とう培養した後、各試験管の全量を同じ番号を記した16本の1.5ml容チューブに移して14,000rpmで1分間(室温)遠心分離した。以下、16本の各1.5ml容チューブについて行った操作を示す。上清をアスピレーターで除去し、STET[0.1M NaCl，10mM Tris-HCl(pH8.0)，1mM EDTA(pH8.0)，5% TritonX-100]350μlを加えて混和し、沈殿を懸濁した。10mM Tris-HCl(pH8.0)に10mg/mlになるよう調製したリゾチーム溶液を25μl加えて3秒間撹拌し、40秒間煮沸した後氷中で5分間冷却した。14,000rpmで10分間(室温)遠心分離し、沈殿を滅菌した爪楊枝を用いて除去し、残った上清に3M酢酸ナトリウム(pH5.2)40μlとイソプロパノール420μlを加えて撹拌した後、室温で5分間静置した。これを14,000rpmで5分間(4℃)遠心分離し、上清を丁寧に取り除いた。得られた沈殿に500μlの70％エタノールを加え、14,000rpmで5分間(4℃)遠心分離後、上清を除去し、沈殿を減圧乾燥した。これにRNaseが20μg/mlとなるよう調整したTEを50μl添加して懸濁し、37℃で30分間静置してプラスミド溶液とした。 Prepare 16 test tubes containing 2 ml of LB culture solution [1% tryptone, 0.5% yeast extract, 1% NaCl] for small-scale culture of transformed colonies. After autoclaving, ampicillin diluted 10-fold to this [ 25 mg / ml] was added to each tube. The colony was scraped with the tip of a sterilized toothpick and brought into contact with the LMA plate to inoculate a part of E. coli to prepare a master plate, and the toothpick was directly put into a test tube containing LB culture solution. The master plate was distinguished by assigning a number for each colony, allowed to stand at 37 ° C. overnight, sealed with vinyl tape, and stored at 4 ° C. The test tube containing the LB culture medium is also given the number corresponding to the master plate, tilted and incubated overnight at 37 ° C, and then 16 1.5ml tubes with the same number for each test tube. And centrifuged at 14,000 rpm for 1 minute (room temperature). The operations performed on each of the 16 1.5 ml tubes are shown below. The supernatant was removed with an aspirator, and STET [0.1 M NaCl, 10 mM Tris-HCl (pH 8.0), 1 mM EDTA (pH 8.0), 5% TritonX-100] was added and mixed to suspend the precipitate. . 25 μl of lysozyme solution prepared to 10 mg / ml in 10 mM Tris-HCl (pH 8.0) was added, stirred for 3 seconds, boiled for 40 seconds and then cooled in ice for 5 minutes. Centrifuge at 14,000 rpm for 10 minutes (room temperature), remove the precipitate using a sterilized toothpick, add 40 μl of 3M sodium acetate (pH 5.2) and 420 μl of isopropanol to the remaining supernatant, and then stir at room temperature for 5 minutes. Let stand for a minute. This was centrifuged at 14,000 rpm for 5 minutes (4 ° C.), and the supernatant was carefully removed. 500 μl of 70% ethanol was added to the resulting precipitate, centrifuged at 14,000 rpm for 5 minutes (4 ° C.), the supernatant was removed, and the precipitate was dried under reduced pressure. To this, 50 μl of TE adjusted to 20 μg / ml of RNase was added and suspended, and allowed to stand at 37 ° C. for 30 minutes to obtain a plasmid solution.
スモールスケール培養により得たプラスミドにFT遺伝子が導入されているかどうかを制限酵素で処理して確認した。プラスミド溶液2μl、10×K Buffer(TaKaRa)1μl、滅菌水6.8μl、XhoＩ(TaKaRa)0.2μlを新たな1.5ml容チューブに移して混合し、37℃で2時間静置した。その後、90μlの滅菌水、50μlのTE飽和フェノール、50μlのクロロホルムを順に加え、ボルテックスミキサーで30秒間撹拌し、14,000rpmで5分間(4℃)遠心分離した。上清100μlに等量のクロロホルムを加え、ボルテックスミキサーで30秒間撹拌した後、14,000rpmで5分間(4℃)遠心分離し、この上清100μlを別の1.5ml容チューブに移した。その上清に10μlの3M 酢酸ナトリウム(pH5.2)、300μlの99％エタノールを加え、十分に撹拌した後、-80℃で30分間静置した。静置後、14,000rpmで10分間(4℃)遠心分離し、得られた沈殿に70％エタノール500μlを加えて14,000rpmで5分間(4℃)遠心分離した。上清を捨て、沈殿を減圧乾燥し、10μlの滅菌水に懸濁した。引き続きこの溶液に10×T Buffer(TaKaRa)を2μl、0.1％BSA(TaKaRa)を2μl、滅菌水を5.6μl、SmaＩ(TaKaRa)を0.4μl混合し、25℃で2時間静置した。静置後、XhoＩとSmaＩで処理したプラスミド溶液のうち9μlに10×Loading buffer 1μlを加え1％アガロースゲルのウェルにアプライして電気泳動した。泳動の結果、PCRにより得られたFT遺伝子と同じサイズの断片がプラスミドから切り出されているサンプルの番号を控え、次のラージスケール培養にその番号の記されたコロニーを用いた。 It was confirmed by treating with a restriction enzyme whether the FT gene was introduced into the plasmid obtained by the small scale culture. 2 μl of the plasmid solution, 1 μl of 10 × K Buffer (TaKaRa), 6.8 μl of sterilized water and 0.2 μl of XhoI (TaKaRa) were transferred to a new 1.5 ml tube, mixed, and allowed to stand at 37 ° C. for 2 hours. Thereafter, 90 μl of sterilized water, 50 μl of TE saturated phenol, and 50 μl of chloroform were sequentially added, and the mixture was stirred with a vortex mixer for 30 seconds and centrifuged at 14,000 rpm for 5 minutes (4 ° C.). An equal volume of chloroform was added to 100 μl of the supernatant, stirred for 30 seconds with a vortex mixer, and then centrifuged at 14,000 rpm for 5 minutes (4 ° C.), and 100 μl of this supernatant was transferred to another 1.5 ml tube. 10 μl of 3M sodium acetate (pH 5.2) and 300 μl of 99% ethanol were added to the supernatant, and after stirring well, the mixture was allowed to stand at −80 ° C. for 30 minutes. After standing, it was centrifuged at 14,000 rpm for 10 minutes (4 ° C.), and 500 μl of 70% ethanol was added to the resulting precipitate, followed by centrifugation at 14,000 rpm for 5 minutes (4 ° C.). The supernatant was discarded and the precipitate was dried under reduced pressure and suspended in 10 μl of sterile water. Subsequently, 2 μl of 10 × T Buffer (TaKaRa), 2 μl of 0.1% BSA (TaKaRa), 5.6 μl of sterilized water, and 0.4 μl of SmaI (TaKaRa) were mixed with this solution and allowed to stand at 25 ° C. for 2 hours. After standing, 10 μl of loading buffer (1 μl) was added to 9 μl of the plasmid solution treated with XhoI and SmaI, and applied to a well of a 1% agarose gel for electrophoresis. As a result of electrophoresis, the number of the sample in which a fragment having the same size as the FT gene obtained by PCR was excised from the plasmid was recorded, and the colony with that number was used for the next large-scale culture.
ラージスケール培養には200μlのアンピシリンを加えた100mlのLB培養液とQIAGEN Plasmid Midi Kit(100)(QIAGEN)を用いて行った。マスタープレートから単一コロニーを爪楊枝で採取し、これをLB培養液の入った坂口フラスコに入れ37℃で12〜16時間振とう培養し、培養後ファルコンチューブに培養液50mlを移して7,500rpmで15分間(4℃)遠心分離した。上清を捨て、残りの50mlも同じように遠心分離し、その後ペレットをRNaseの入った4mlのBuffer P1に懸濁して4mlのBuffer P2を添加後、4〜6回転倒させ十分に混和し５分間室温で静置した。冷却したBuffer P3を4ml添加して4〜6回転倒させ混和した後、15分間氷上でインキュベーションした。続いて50ml容の遠心管に溶液を移し、13,000rpm(日立RPR16ローター)で30分間(4℃)遠心分離し、プラスミドDNAを含む上清を回収した。この上清を再度13,000rpmで15分間 (4℃)遠心分離した。カラムに4mlのBuffer QBTを加えカラムが空になるまで自然落下させ平衡化し、遠心分離で得られた上清をこのカラムに添加して樹脂に浸透させた。カラムを10mlのBuffer QCで2回洗浄し、5mlのBuffer QFでDNAを新たなファルコンチューブに溶出した。溶出したDNA溶液にイソプロパノール3.5mlを添加し混和した後、COREXチューブへ入れ直ちに11,000rpmで30分間(4℃)遠心分離(日立RPR16ローター)し、上清を素早くデカンテーションで除去した。沈殿したDNAを70％エタノール2mlで洗浄し、11,000rpmで10分間(4℃)遠心分離し、上清を取り除いて沈殿したDNAを減圧乾燥した後、TE100μlに溶解した。分光光度計（ND-1000 v3.1.2）を用いてDNAの濃度を1μg/μlに調製し、pEALSR2L5R5にFT遺伝子を導入した感染性cDNAクローンpEALSR2L5R5FTを得た。
(2) 感染性cDNAクローンのウイルス化
(1)の方法に従って、ラージスケール培養から精製したALSV RNA1 の感染性cDNAクローンであるpEALSR1を1μg/μlに調製した。増殖宿主のChenopodium quinoa（以下キノア）の葉にカーボランダムをふりかけ、pEALSR1(1μg/μl)とpEALSR2L5R5FT(1μg/μl)を等量ずつ混ぜ合わせたDNA溶液をこれら1葉当たり8μlずつ接種しウイルス化を行うことでFT遺伝子を発現するALSVベクター（FT-ALSV）を得た。FT-ALSV感染により退緑症状が現れたキノア葉をサンプリングし、以後の操作に用いた。
(3) ウイルスの濃縮
FT遺伝子を連結したALSV（FT-ALSV）の濃縮は以下の手順で行った。まず、FT-ALSVをChenopodium quinoa（以下キノア）に接種後、7〜10日の接種葉と病徴の現れた上葉をサンプリングした。この感染葉100gに対し、300mlの抽出緩衝液 [0.1M Tris-HCl（pH7.8），0.1M NaCl，5mM MgCl2]と3mlのメルカプトエタノールを加えてワーリングブレンダーで磨砕した。磨砕液を2重ガーゼでろ過し、9,000rpmで10分間（4℃）遠心分離（日立RPR12-2ローター）した。得られた溶液に、ベントナイト溶液（40mg/ml）を攪拌しながら静かに加え、9,000rpmで10分間（4℃）遠心分離した。上清が透明な黄色になるまでこの作業を繰り返し清澄化した。次に、上清にポリエチレングリコールを8％になるように加え、氷中で1時間攪拌した。9,000rpmで10分間（4℃）遠心分離した後、沈殿を20mlの抽出緩衝液に溶解した。続いて10mlのクロロホルムを加えて15分間（4℃）攪拌した。9,000rpmで10分間（4℃）遠心分離（日立RPR16ローター）後、上清を45,000rpmで1.5時間（4℃）遠心分離（日立RP65ローター）した。沈殿に1mlの抽出緩衝液を加えて十分に懸濁した後、9,000rpmで10分間（4℃）遠心分離しこの上清を濃縮FT-ALSVとした。
(4) RNAの抽出
濃縮FT-ALSVからRNAを抽出し、マイクロキャリアの調製に用いた。濃縮FT-ALSVからのRNAの抽出は以下の手順で行った。濃縮FT-ALSV 50μlに滅菌水150μlを加え撹拌した後、水飽和フェノールとクロロホルムを100μlずつ加えてボルテックスミキサーで十分に攪拌した。14,000rpmで5分間(4℃)遠心分離後、上清200μlを新しい1.5ml容チューブに移し、20μlの3M 酢酸ナトリウム(pH5.2)、500μlの99％エタノールを加え、十分に撹拌した後、-80℃で15分間静置した。14,000rpmで15分間(4℃)遠心分離して得られた沈殿に70％エタノール1mlを加えて14,000rpmで5分間(4℃)遠心分離した。上清を捨て、15μlの滅菌水に懸濁した。分光光度計(ND-1000 v3.1.2)で得られた溶液を1μg/μlに調整し、これをRNA溶液とした。
(5)パーティクルガン接種(Biolistic PDS-1000/He Particle Delively System（Bio-Rad
）)
マイクロキャリア（RNA;3μg/金粒子;0.4mg/shot）の調製は以下の手順で行った。まず、1.5ml容チューブに金粒子(0.6μm)を2.4mg量り取り、滅菌水100μlを加えボルテックスミキサーで十分に混和した。超音波洗浄機にチューブを入れ、5分間ソニケーションした。ボルテックスミキサーにチューブをセットし撹拌しながら18μlのRNA溶液（1μg/μl）を静かに加えた。同様に、5M 酢酸アンモニウムを11.8μl、続いてイソプロパノールを259.6μl静かに加えた。しばらく撹拌した後、−20℃で1時間以上静置した。上清を取り除き金粒子の沈殿をボルテックスミキサーで一瞬撹拌した。金粒子の沈殿に1mlの100%エタノールを加え、沈殿を崩さないように静かに振盪し、その後上清を取り除いた。この作業を4回繰り返した後に60μlの100%エタノールに金粒子を懸濁した。懸濁液10μlをマイクロキャリアの中心に約1cmに塗り広げ十分乾燥させた後に、パーティクルガンを用いて植物への導入を行った。パーティクルガンを用いた接種試験には発根して間もないリンゴの種子4〜6個体を1試験区として供試した。種皮をメスで取り除きシャーレに同心円状に並べ、その子葉にヘリウム圧1100psiでBiolistic PDS-1000/He Particle Delively System（Bio-Rad）を用いて1試験区あたり4shot行った。
(6) パーティクルガン接種(Helios Gene Gun System（Bio-Rad）)
マイクロキャリア（RNA;3μg/金粒子;0.4mg/shot）の調製は以下の手順で行った。まず、1.5ml容チューブに金粒子(1.0μm)を7.2mg量り取り、滅菌水100μlを加えボルテックスミキサーで十分に混和した。超音波洗浄機にチューブを入れ、5分間ソニケーションした。ボルテックスミキサーにチューブをセットし撹拌しながら54μlのRNA溶液（1μg/μl）を静かに加えた。同様に、5M 酢酸アンモニウムを15.4μl、続いてイソプロパノールを338.8μl静かに加えた。しばらく撹拌した後、−20℃で1時間以上静置した。上清を取り除き金粒子の沈殿をボルテックスミキサーで一瞬撹拌した。金粒子の沈殿に1mlの100%エタノールを加え、沈殿を崩さないように静かに振盪し、その後上清を取り除いた。この作業を4回繰り返した後に1080μlの100%エタノールに金粒子を懸濁し、ゴールドコートチューブの調製に使用した。チュービングプレップステーション（BIO-RAD）にゴールドコートチューブ（BIO-RAD）をセットし、純窒素ガスを20分間通してゴールドコートチューブの内部を完全に乾燥させた。続いて金粒子の懸濁液を、均一になるようゴールドコートチューブ内に充填し、5分間放置して金粒子をゴールドコートチューブに沈着させた後、上清の99.5%エタノールをゴールドコートチューブ内から取り除いた。続いてチュービングプレップステーションを回転させ、金粒子をゴールドコートチューブ内部に均一に拡散させながらゴールドコートチューブ内に純チッソガスを通し、金粒子を完全に乾燥させた。続いてゴールドコートチューブを、チューブカッター（BIO-RAD）を用いて18個に裁断し、パーティクルガンを用いて植物への導入を行った。発根して間もないリンゴ種子の種皮をメスで取り除き、その子葉に接種した。接種はヘリウム圧220psiでHelios Gene Gun System（Bio-Rad）を用いて1個体あたり4shot行った。
(7) 接種個体の育成
接種個体は湿度を保ち、遮光条件下で2〜3日静置した後に、徐々に外気に馴化させ、その後培養土に移植し、培養室(25℃、明期16時間-暗期8時間)で育成した。
(8) 花粉発芽試験
花粉の発芽率はショ糖17％を含む1％寒天培地上に花粉を散布し、培養室に12時間以上静置後、一花につき120粒以上光学顕微鏡下で観察し、花粉管が伸長している花粉の割合から求めた。
2．結果
濃縮FT-ALSVから抽出したRNAを発根直後のリンゴ実生の子葉にパーティクルガン法を用いて接種することにより、接種した20個体中20個体においてFT-ALSVの全身感染が確認できた。したがって、本法を用いることにより非常に高い効率でALSVベクターをリンゴに感染させられることができることが明らかとなった。また、FT-ALSVの感染が確認された20個体中8個体で、接種後1.5ヶ月から3ヶ月の間に、図１に示したように開花が認められた。開花した各個体から花粉を採取し発芽試験を行った結果、いずれの個体の花粉も発芽能力を有し、高いものでは図2に示したように、発芽率は80％近かった。すなわち、FT-ALSVを利用することで通常6年から12年を要するリンゴの開花までの期間を大幅に短縮できるとともに、FT-ALSV感染により早期開花したリンゴを花粉親に用いることが可能であると考えられた。
Large-scale culture was performed using 100 ml of LB medium supplemented with 200 μl of ampicillin and QIAGEN Plasmid Midi Kit (100) (QIAGEN). Pick a single colony from the master plate with a toothpick, place it in a Sakaguchi flask containing LB culture solution, shake culture at 37 ° C for 12-16 hours, transfer 50 ml of the culture solution to a falcon tube after incubation, and transfer at 7,500 rpm. Centrifuge for 15 minutes (4 ° C). Discard the supernatant and centrifuge the remaining 50 ml in the same way, then suspend the pellet in 4 ml Buffer P1 containing RNase, add 4 ml Buffer P2, and invert 4-6 times to mix thoroughly. Let stand at room temperature for minutes. 4 ml of cooled Buffer P3 was added and mixed by inverting 4-6 times, and then incubated on ice for 15 minutes. Subsequently, the solution was transferred to a 50 ml centrifuge tube and centrifuged at 13,000 rpm (Hitachi RPR16 rotor) for 30 minutes (4 ° C.), and the supernatant containing the plasmid DNA was recovered. The supernatant was again centrifuged at 13,000 rpm for 15 minutes (4 ° C.). 4 ml of Buffer QBT was added to the column and allowed to drop and equilibrate until the column was empty, and the supernatant obtained by centrifugation was added to this column and allowed to permeate the resin. The column was washed twice with 10 ml Buffer QC, and DNA was eluted into a new falcon tube with 5 ml Buffer QF. After adding 3.5 ml of isopropanol to the eluted DNA solution and mixing, it was placed in a COREX tube, immediately centrifuged at 11,000 rpm for 30 minutes (4 ° C.) (Hitachi RPR16 rotor), and the supernatant was quickly removed by decantation. The precipitated DNA was washed with 2 ml of 70% ethanol, centrifuged at 11,000 rpm for 10 minutes (4 ° C.), the supernatant was removed, the precipitated DNA was dried under reduced pressure, and then dissolved in 100 μl of TE. The concentration of DNA was adjusted to 1 μg / μl using a spectrophotometer (ND-1000 v3.1.2) to obtain an infectious cDNA clone pEALSR2L5R5FT in which the FT gene was introduced into pEALSR2L5R5.
(2) Viralization of infectious cDNA clones
According to the method of (1), pEALSR1, which is an infectious cDNA clone of ALSV RNA1 purified from large scale culture, was prepared at 1 μg / μl. Sprinkle carborundum on the leaves of the growth host Chenopodium quinoa (hereinafter referred to as quinoa), and inoculate 8 μl of each DNA solution containing pEALSR1 (1 μg / μl) and pEALSR2L5R5FT (1 μg / μl) in an equal amount. To obtain an ALSV vector (FT-ALSV) expressing the FT gene. The quinoa leaves that showed deciduous symptoms due to FT-ALSV infection were sampled and used for subsequent operations.
(3) Virus concentration
Concentration of ALSV linked with FT gene (FT-ALSV) was carried out by the following procedure. First, after inoculating FT-ALSV into Chenopodium quinoa (hereinafter referred to as quinoa), 7 to 10 days of inoculated leaves and upper leaves showing disease symptoms were sampled. To 100 g of the infected leaves, 300 ml of extraction buffer [0.1 M Tris-HCl (pH 7.8), 0.1 M NaCl, 5 mM MgCl2] and 3 ml of mercaptoethanol were added and ground with a Waring blender. The ground liquid was filtered through double gauze and centrifuged (Hitachi RPR12-2 rotor) at 9,000 rpm for 10 minutes (4 ° C.). To the obtained solution, a bentonite solution (40 mg / ml) was gently added while stirring, and centrifuged at 9,000 rpm for 10 minutes (4 ° C.). This operation was repeated and clarified until the supernatant became clear yellow. Next, polyethylene glycol was added to the supernatant to 8%, and the mixture was stirred in ice for 1 hour. After centrifugation at 9,000 rpm for 10 minutes (4 ° C.), the precipitate was dissolved in 20 ml of extraction buffer. Subsequently, 10 ml of chloroform was added and stirred for 15 minutes (4 ° C.). After centrifugation at 9,000 rpm for 10 minutes (4 ° C.) (Hitachi RPR16 rotor), the supernatant was centrifuged at 45,000 rpm for 1.5 hours (4 ° C.) (Hitachi RP65 rotor). After 1 ml of extraction buffer was added to the precipitate and suspended sufficiently, it was centrifuged at 9,000 rpm for 10 minutes (4 ° C.), and this supernatant was used as concentrated FT-ALSV.
(4) RNA extraction RNA was extracted from the concentrated FT-ALSV and used to prepare microcarriers. RNA was extracted from the concentrated FT-ALSV by the following procedure. After 150 μl of sterilized water was added to 50 μl of concentrated FT-ALSV and stirred, 100 μl each of water-saturated phenol and chloroform were added and stirred thoroughly with a vortex mixer. After centrifugation at 14,000 rpm for 5 minutes (4 ° C), transfer 200 μl of the supernatant to a new 1.5 ml tube, add 20 μl of 3M sodium acetate (pH 5.2), 500 μl of 99% ethanol, and stir well. It was allowed to stand at -80 ° C for 15 minutes. To the precipitate obtained by centrifugation at 14,000 rpm for 15 minutes (4 ° C.), 1 ml of 70% ethanol was added and centrifuged at 14,000 rpm for 5 minutes (4 ° C.). The supernatant was discarded and suspended in 15 μl of sterile water. The solution obtained with a spectrophotometer (ND-1000 v3.1.2) was adjusted to 1 μg / μl, and this was used as an RNA solution.
(5) Particle gun inoculation (Biolistic PDS-1000 / He Particle Delively System (Bio-Rad
))
The microcarrier (RNA; 3 μg / gold particle; 0.4 mg / shot) was prepared by the following procedure. First, 2.4 mg of gold particles (0.6 μm) was weighed into a 1.5 ml tube, 100 μl of sterilized water was added, and the mixture was thoroughly mixed with a vortex mixer. The tube was placed in an ultrasonic cleaner and sonicated for 5 minutes. The tube was set in a vortex mixer, and 18 μl of RNA solution (1 μg / μl) was gently added while stirring. Similarly, 11.8 μl of 5M ammonium acetate was added gently followed by 259.6 μl of isopropanol. After stirring for a while, the mixture was allowed to stand at -20 ° C for 1 hour or longer. The supernatant was removed and the gold particle precipitate was stirred for a moment with a vortex mixer. To the gold particle precipitate, 1 ml of 100% ethanol was added, and gently shaken so as not to break the precipitate, and then the supernatant was removed. After this operation was repeated 4 times, the gold particles were suspended in 60 μl of 100% ethanol. After 10 μl of the suspension was applied to the center of the microcarrier to about 1 cm and sufficiently dried, it was introduced into plants using a particle gun. In an inoculation test using a particle gun, 4 to 6 apple seeds that had just been rooted were used as one test plot. The seed coat was removed with a scalpel and arranged concentrically in a petri dish, and 4 shots per test group were performed on the cotyledons using a Biolistic PDS-1000 / He Particle Delively System (Bio-Rad) at a helium pressure of 1100 psi.
(6) Particle gun inoculation (Helios Gene Gun System (Bio-Rad))
The microcarrier (RNA; 3 μg / gold particle; 0.4 mg / shot) was prepared by the following procedure. First, 7.2 mg of gold particles (1.0 μm) was weighed into a 1.5 ml tube, 100 μl of sterilized water was added, and the mixture was thoroughly mixed with a vortex mixer. The tube was placed in an ultrasonic cleaner and sonicated for 5 minutes. The tube was set in a vortex mixer, and 54 μl of RNA solution (1 μg / μl) was gently added while stirring. Similarly, 15.4 μl of 5M ammonium acetate was added gently followed by 338.8 μl of isopropanol. After stirring for a while, the mixture was allowed to stand at -20 ° C for 1 hour or longer. The supernatant was removed and the gold particle precipitate was stirred for a moment with a vortex mixer. To the gold particle precipitate, 1 ml of 100% ethanol was added, and gently shaken so as not to break the precipitate, and then the supernatant was removed. After this operation was repeated four times, gold particles were suspended in 1080 μl of 100% ethanol and used for the preparation of a gold-coated tube. A gold coated tube (BIO-RAD) was set in a tubing prep station (BIO-RAD), and pure nitrogen gas was passed for 20 minutes to completely dry the inside of the gold coated tube. Subsequently, the gold particle suspension is filled uniformly in the gold coat tube and left for 5 minutes to deposit the gold particles in the gold coat tube. Then, 99.5% ethanol of the supernatant is placed in the gold coat tube. Removed from. Subsequently, the tubing prep station was rotated, and pure gold gas was passed through the gold coat tube while uniformly diffusing the gold particles into the gold coat tube, thereby completely drying the gold particles. Subsequently, the gold coated tube was cut into 18 pieces using a tube cutter (BIO-RAD), and introduced into a plant using a particle gun. The seed coat of the apple seed that had just been rooted was removed with a scalpel and inoculated into its cotyledons. Inoculation was performed for 4 shots per individual using a Helios Gene Gun System (Bio-Rad) at a helium pressure of 220 psi.
(7) Breeding of the inoculated individuals The inoculated individuals are kept humidity and allowed to stand for 2 to 3 days under light-shielded conditions, then gradually acclimatized to the outside air, then transplanted to the culture soil, and cultured in the culture room (25 ° C, light period 16 Time-dark period 8 hours).
(8) Pollen germination test The pollen germination rate was determined by spraying pollen on a 1% agar medium containing 17% sucrose, leaving it in the culture room for 12 hours or more, and observing 120 or more per flower under an optical microscope. From the ratio of pollen in which the pollen tube is elongated.
2． Results By inoculating RNA extracted from concentrated FT-ALSV into the cotyledons of apple seedlings immediately after rooting using the particle gun method, systemic infection of FT-ALSV was confirmed in 20 of 20 inoculated individuals. Therefore, it has been clarified that by using this method, apples can be infected with the ALSV vector with very high efficiency. In addition, 8 out of 20 individuals confirmed to be infected with FT-ALSV showed flowering as shown in FIG. 1 within 1.5 to 3 months after inoculation. As a result of collecting pollen from each flowered individual and conducting a germination test, the pollen of any individual had a germination ability, and as shown in FIG. 2, the germination rate was close to 80%. In other words, the use of FT-ALSV can significantly shorten the period until apples normally take 6 to 12 years, and it is possible to use apples that flowered early due to FT-ALSV infection as pollen parents. It was considered.
以上詳しく説明したように、本願発明によって、リンゴの開花時期を大幅に早めることにより短期間でリンゴの花粉を取得することが可能となる。これは、リンゴの品種改良に大いに資するものである。 As explained in detail above, according to the present invention, apple pollen can be obtained in a short period of time by greatly advancing the flowering time of apples. This greatly contributes to the improvement of apple varieties.
Claims (1)
Including inoculating the cotyledons of apple seedlings immediately after rooting using a particle gun method with viral RNA concentrated from a recombinant apple small spherical latent virus (FT-ALSV) expressing the Arabidopsis FT gene A method for obtaining pollen of apples, comprising: flowering 1.5 to 3 months after sowing, and collecting pollen having germination ability from the flower.
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