JP4534034B2 - Agrobacterium with improved efficiency of gene introduction into plants and method for producing the same - Google Patents
Agrobacterium with improved efficiency of gene introduction into plants and method for producing the same Download PDFInfo
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- JP4534034B2 JP4534034B2 JP2004133070A JP2004133070A JP4534034B2 JP 4534034 B2 JP4534034 B2 JP 4534034B2 JP 2004133070 A JP2004133070 A JP 2004133070A JP 2004133070 A JP2004133070 A JP 2004133070A JP 4534034 B2 JP4534034 B2 JP 4534034B2
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Description
本発明は、アグロバクテリウム菌に、当該アグロバクテリウム菌により外来遺伝が導入される植物のエチレンの生産を抑制する能力を付与することにより、特にACCデアミナーゼ遺伝子あるいはリゾビトキシン生合成系遺伝子を導入することにより、植物へ遺伝子を導入する効率が向上したアグロバクテリウム菌およびその作製方法に関する。 The present invention introduces, in particular, an ACC deaminase gene or a lysobitoxin biosynthetic gene by imparting to Agrobacterium the ability to suppress the production of ethylene in plants into which foreign genes are introduced by the Agrobacterium. The present invention relates to an Agrobacterium having improved efficiency of introducing a gene into a plant and a method for producing the same.
現在、トウモロコシ、ダイズ、ワタ、ナタネなどの遺伝子組換え作物が商品化されている。栽培の中心は北米や南米であるが、中国やインドなど栽培地域が世界的な広がりをみせている。また、多くの種類の植物で遺伝子組換え植物の開発が実用化に向けて行われている。遺伝子組換え植物の作製法には、遺伝子銃を使用した直接法とアグロバクテリウム菌を使用した間接法が開発されているが、後者の方法が主要な方法として使用されている。なおアグロバクテリウム菌は根粒菌と近い土壌細菌であり、植物細胞をアグロバクテリウム菌に感染させることにより細菌の遺伝子がその植物細胞に転位する。よってアグロバクテリウム菌により仲介された遺伝子導入が可能となる。 Currently, genetically modified crops such as corn, soybeans, cotton, rapeseed are commercialized. The center of cultivation is North America and South America, but growing areas such as China and India are spreading worldwide. In addition, genetically modified plants are being developed for practical use in many types of plants. As a method for producing a transgenic plant, a direct method using a gene gun and an indirect method using Agrobacterium have been developed, but the latter method is used as a main method. Agrobacterium is a soil bacterium similar to rhizobia, and the bacterial gene is transferred to the plant cell by infecting the plant cell with Agrobacterium. Therefore, gene transfer mediated by Agrobacterium is possible.
しかしながら、現在までに開発されているアグロバクテリウム法は、遺伝子を導入しようとする植物の種類や品種の違いによって遺伝子導入効率に差がある。そのまたその遺伝子導入効率は極めて悪いためにしばしば問題を生じる。例えば、トマトやタバコ(Hanson et al., 1999)、イネ(Hiei et al., 1994)などでは高い効率が得られるが、コムギ(Cheng et al.,1997)、ホップ(Horlemann et al., 2003)、ナタネ(Cardoza et al., 2003)、メロン(Ezura et al., 2000)など商業的に重要な栽培植物品種への遺伝子導入の効率は低いままである。 However, the Agrobacterium method that has been developed so far has a difference in gene transfer efficiency depending on the type and variety of plants to which the gene is to be transferred. In addition, its gene transfer efficiency is extremely poor, which often causes problems. For example, tomato, tobacco (Hanson et al., 1999), rice (Hiei et al., 1994), etc. have high efficiency, but wheat (Cheng et al., 1997), hop (Horlemann et al., 2003) ), Rapeseed (Cardoza et al., 2003), melon (Ezura et al., 2000), etc., the efficiency of gene transfer to commercially important cultivated plant varieties remains low.
そこで、植物細胞に遺伝子を導入する際に使用するアグロバクテリウム懸濁液にアグロバクテリウム菌の植物細胞への感染を促進する物質を添加することにより、遺伝子導入効率を促進する研究が行われている。そして既にいくつかの促進物質が見つかっており、効率的な組換え植物体の作製に利用されている。 Therefore, studies have been conducted to increase gene transfer efficiency by adding substances that promote the infection of Agrobacterium to plant cells to the Agrobacterium suspension used when introducing genes into plant cells. ing. Several promoters have already been found and are used for the production of efficient recombinant plants.
例えば、アセトシリンゴンは、アグロバクテリウム菌の植物への感染を促進する物質であるために、ワタやトマトなどへのアグロバクテリウム法による遺伝子組換え体の作製に使用されている（Sunikumar and Rathore, 2001, Molecular Breeding, 8:37-52）。 For example, since acetosyringone is a substance that promotes plant infection with Agrobacterium, it is used for the production of genetically modified organisms such as cotton and tomato by the Agrobacterium method (Sunikumar and Rathore, 2001, Molecular Breeding, 8: 37-52).
遺伝子組換え技術を使った植物の分子育種では、遺伝子導入の対象となる植物の種類や品種に効率的に遺伝子導入できることが極めて重要である。しかしながら、植物への遺伝子導入法として主に使われているアグロバクテリウム法は、対象となる植物の種類や品種によって遺伝子導入効率が低く、植物分子育種の大きな問題である。世界的な広がりを見せている植物分子育種を効果的に進めるには、植物の種類や品種に依存しない効率的なアグロバクテリウム菌による遺伝子導入法を開発する必要が存在した。このような理由から、本発明者等は、これまで植物とアグロバクテリウム菌の相互作用に関する研究を続けてきている。 In molecular breeding of plants using gene recombination technology, it is extremely important that genes can be efficiently introduced into the types and varieties of plants to which genes are introduced. However, the Agrobacterium method, which is mainly used as a method for gene transfer into plants, has a low gene transfer efficiency depending on the type and variety of the target plant, and is a big problem in plant molecular breeding. In order to effectively promote the molecular breeding of plants that are spreading globally, it was necessary to develop an efficient gene transfer method using Agrobacterium that does not depend on the type or variety of plants. For these reasons, the present inventors have continued research on the interaction between plants and Agrobacterium.
本発明はこのような状況に鑑みてなされたものであり、その目的は、広く植物において利用することが可能であって、植物へ遺伝子を導入する効率が向上したアグロバクテリウム菌およびその作製方法を提供することにある。 The present invention has been made in view of such circumstances, and its object is to be widely used in plants, and an Agrobacterium having improved efficiency of introducing a gene into a plant and a method for producing the same. Is to provide.
本発明者等は、これまでにアグロバクテリウム法によるメロンへの遺伝子導入に際して、メロン細胞がエチレンを生成してアグロバクテリウム菌から植物細胞への遺伝子導入を低下させていること、逆にエチレン生合成阻害剤を加えてメロン細胞のエチレン生成を抑制するとアグロバクテリウム菌からの遺伝子導入効率が向上することを明らかにしている（Ezuraら、2000, Plant Breeding,119:75-79）。 The present inventors have heretofore confirmed that, when a gene is introduced into melon by the Agrobacterium method, melon cells have produced ethylene to reduce gene introduction from Agrobacterium to plant cells. It has been clarified that the efficiency of gene transfer from Agrobacterium is improved by adding biosynthesis inhibitors to suppress ethylene production in melon cells (Ezura et al., 2000, Plant Breeding, 119: 75-79).
そこで本発明者等は、上記課題を解決すべく、植物のエチレン生合成を阻害する物質を生産する能力を付与したアグロバクテリウム菌の作製を試みた結果、遂に植物のエチレン生合成阻害物質であるACCデアミナーゼを生産する能力を有するアグロバクテリウム菌の作製に成功した。 In order to solve the above problems, the present inventors have attempted to produce Agrobacterium having the ability to produce a substance that inhibits plant ethylene biosynthesis. Agrobacterium having the ability to produce an ACC deaminase was successfully produced.
作製したアグロバウテリウム菌の能力を検討した結果、植物が生産するエチレンの前駆物質であるACCを分解することができた。微生物感染により単子葉及び双子葉を含む植物がエチレンを生成する現象は公知の事実であり、本発明者等は、作製したアグロバクテリム菌が広く植物細胞からのエチレン生成を抑制し、遺伝子を効率的に導入できることを見出した。よって本発明は上記課題を解決するために行われたものであり、植物への遺伝子導入能力を向上させたアグロバクテリウム菌およびその作製方法に関するものである。 As a result of examining the ability of the produced Agrobacterium, it was possible to decompose ACC, which is a precursor of ethylene produced by plants. It is a known fact that plants including monocotyledonous and dicotyledonous plants produce ethylene due to microbial infection, and the present inventors have widely suppressed the production of ethylene from plant cells by the produced Agrobacterium. We found that it can be introduced efficiently. Therefore, the present invention has been made to solve the above-described problems, and relates to an Agrobacterium having an improved ability to introduce a gene into a plant and a method for producing the same.
一つの観点において本発明は、アグロバクテリウム菌に、ＡＣＣデアミナーゼ遺伝子をサブクローニングすることにより、当該アグロバクテリウム菌により外来遺伝子が導入される植物のエチレンの生産を抑制する能力を付与して、植物へ外来遺伝子を導入する効率が向上したアグロバクテリウム菌を作製する方法である。 In one aspect, the present invention provides Agrobacterium with the ability to suppress the production of ethylene in a plant into which a foreign gene is introduced by subcloning the ACC deaminase gene by the Agrobacterium, This is a method for producing an Agrobacterium having improved efficiency in introducing a foreign gene into a cell .
更に一つの観点において本発明は、アグロバクテリウム菌に、ＡＣＣデアミナーゼ遺伝子をサブクローニングすることにより、当該アグロバクテリウム菌により外来遺伝子が導入される植物のエチレンの生産を抑制する能力を付与して、植物へ外来遺伝子を導入する効率が向上したアグロバクテリウム菌である。 Furthermore, in one aspect, the present invention provides Agrobacterium with the ability to suppress the production of ethylene in a plant into which a foreign gene is introduced by subcloning the ACC deaminase gene. It is an Agrobacterium with improved efficiency of introducing foreign genes into plants .
更に一つの観点において本発明は、アグロバクテリウム菌に、リゾビトキシン生合成系遺伝子をサブクローニングすることにより、当該アグロバクテリウム菌により外来遺伝子が導入される植物のエチレンの生産を抑制する能力を付与して、植物へ外来遺伝子を導入する効率が向上したアグロバクテリウム菌を作製する方法である。 Furthermore, in one aspect, the present invention provides Agrobacterium with the ability to suppress the ethylene production of plants into which foreign genes are introduced by Agrobacterium by subcloning a lysobitoxin biosynthesis gene. This is a method for producing Agrobacterium having improved efficiency in introducing a foreign gene into a plant .
更に一つの観点において本発明は、アグロバクテリウム菌にリゾビトキシン生合成系遺伝子をサブクローニングすることにより、当該アグロバクテリウム菌により外来遺伝子が導入される植物のエチレンの生産を抑制する能力を付与して、植物へ外来遺伝子を導入する効率が向上したアグロバクテリウム菌である。 Furthermore, in one aspect, the present invention provides the ability to suppress ethylene production in plants into which foreign genes are introduced by the Agrobacterium by subcloning a lysobitoxin biosynthesis gene into Agrobacterium. Agrobacterium with improved efficiency of introducing foreign genes into plants .
本発明により、アグロバクテリウム菌に、当該アグロバクテリウム菌によって外来遺伝子を導入しようとする植物のエチレンの生産を抑制する能力を付与することにより、特にACCデアミナーゼ遺伝子あるいはリゾビトキシン生合成系遺伝子を導入することにより、植物へ遺伝子を導入する効率が向上したアグロバクテリウム菌およびその作製方法が提供された。より具体的には本発明の方法により、ACCデアミナーゼ遺伝子またはリゾビトキシン生合成系遺伝子をアグロバクテリウム菌に導入することにより、植物へ遺伝子を導入する効率を向上させることが可能となった。本発明により、アグロバクテリウム菌を用いた方法によって従来は遺伝子導入ができなかった植物種においても、組換え植物を作出することが可能になると考えられる。 According to the present invention, an ACC deaminase gene or a lysobitoxin biosynthetic gene is introduced to Agrobacterium by giving it the ability to suppress the production of ethylene in plants to which foreign genes are to be introduced by the Agrobacterium. Thus, an Agrobacterium having improved efficiency of introducing a gene into a plant and a method for producing the same were provided. More specifically, the efficiency of gene introduction into plants can be improved by introducing the ACC deaminase gene or lysobitoxin biosynthesis gene into Agrobacterium by the method of the present invention. According to the present invention, it is considered that it is possible to produce a recombinant plant even in a plant species that could not conventionally be introduced by a method using Agrobacterium.
本発明は、遺伝子を導入する効率を向上させたアグロバクテリウム菌およびその作製方法を提供するものであり、本発明の方法で作製された菌は植物の形質転換において広く利用することが可能である。 The present invention provides an Agrobacterium having improved gene introduction efficiency and a method for producing the same, and the fungus produced by the method of the present invention can be widely used in plant transformation. is there.
本発明者等は、アグロバクテリウム菌によりメロンへ遺伝子導入を行なう際のエチレンの影響を検討した結果、アグロバクテリウム菌からメロン細胞への遺伝子導入を細胞自らが生産するエチレンが抑制していること、さらにエチレン生合成を抑制する物質を加え植物細胞からのエチレン生合成を抑制することで遺伝子導入効率が向上することを見い出した（実施例１）。これらの観察から、本発明者等は、アグロバクテリウム菌自身に植物細胞のエチレン生成を抑制する物質を生産する能力を付与すれば、アグロバクテリウムの植物への遺伝子導入能力を向上できると考えた。 As a result of studying the influence of ethylene when a gene is introduced into melon by Agrobacterium, the present inventors have suppressed ethylene introduced by the cell itself from Agrobacterium to melon cells. In addition, it was found that gene introduction efficiency is improved by adding a substance that suppresses ethylene biosynthesis to suppress ethylene biosynthesis from plant cells (Example 1). From these observations, the present inventors believe that if Agrobacterium itself is given the ability to produce a substance that suppresses the production of ethylene in plant cells, the ability of Agrobacterium to introduce genes into plants can be improved. It was.
遺伝子導入能力の向上したアグロバクテリウム菌は、産業上、様々な有用性がある。現在、トウモロコシ、ダイズ、ワタ、ナタネなどの遺伝子組換え作物が商品化されている。栽培の中心は北米や南米であるが、中国やインドなど栽培地域が世界的な広がりをみせている。また、多くの種類の植物で遺伝子組換え植物の開発が実用化に向けて行われている。遺伝子組換え植物の作製法には、アグロバクテリウム菌を使った間接法が主に使用されている。しかしながら、現在までに開発さているアグロバクテリウム法は、遺伝子を導入しようとする植物の種類や品種の違いによって、遺伝子導入効率が極めて悪く、しばしば問題を生じてしまう。 Agrobacterium having improved gene transfer ability has various industrial uses. Currently, genetically modified crops such as corn, soybeans, cotton, rapeseed are commercialized. The center of cultivation is North America and South America, but growing areas such as China and India are spreading worldwide. In addition, genetically modified plants are being developed for practical use in many types of plants. Indirect methods using Agrobacterium are mainly used as methods for producing transgenic plants. However, the Agrobacterium method that has been developed so far has a very poor gene transfer efficiency due to differences in the types and varieties of plants to which genes are to be introduced, and often causes problems.
例えば、トマトやタバコ(Hanson et al., 1999)、イネ(Hiei et al., 1994)などでは高い効率が得られ、コムギ(Cheng et al.,1997)、ホップ(Horlemann et al., 2003)、ナタネ(Cardoza et al., 2002)、メロン(Ezura et al., 2000)など商業的に重要な栽培植物品種への遺伝子導入は低い効率である。本発明で開発したアグロバクテリウム菌を用いれば、従来から遺伝子導入効率が低いとされてきた植物への遺伝子導入を効率的に行なうことが可能となる。そして、遺伝子組換え植物の開発を効率的に行なうことができる。 For example, high efficiency is obtained with tomato, tobacco (Hanson et al., 1999), rice (Hiei et al., 1994), wheat (Cheng et al., 1997), hop (Horlemann et al., 2003). Gene transfer into commercially important cultivated plant varieties such as rapeseed (Cardoza et al., 2002) and melon (Ezura et al., 2000) is of low efficiency. By using the Agrobacterium developed in the present invention, it becomes possible to efficiently introduce a gene into a plant that has been considered to have a low gene introduction efficiency. And it is possible to efficiently develop genetically modified plants.
本発明では、植物細胞のエチレン生成を抑制する物質としてACCデアミナーゼもしくはリゾビトキシンを候補として、アグロバクテリウム菌にこの物質を生産する能力を付与した。ACCデアミナーゼは、エチレンの前駆物質であるACCをα−ケト酪酸とアンモニアに分解することが知られている（Honmaら,1978）。具体的には、根粒菌の一種であるMesorhizobium loti（M.loti）のACCデアミナーゼ遺伝子をアグロバクテリウム菌に導入することで、同能力を付与した。ACCデアミナーゼ遺伝子は、M.lotiに限らずいくつかの微生物がもっている遺伝子であり、本発明に使用するACCデアミナーゼ遺伝子は、M. lotiのACCデアミナーゼ遺伝子に制限されるものではない。 In the present invention, ACC deaminase or lysobitoxin is a candidate as a substance that suppresses the production of ethylene in plant cells, and the ability to produce this substance was given to Agrobacterium. ACC deaminase is known to degrade ACC, an ethylene precursor, into α-ketobutyric acid and ammonia (Honma et al., 1978). Specifically, the same ability was imparted by introducing the ACC deaminase gene of Mesorhizobium loti (M.loti), a kind of rhizobia, into Agrobacterium. The ACC deaminase gene is not limited to M. loti, but is a gene possessed by several microorganisms. The ACC deaminase gene used in the present invention is not limited to the ACC deaminase gene of M. loti.
リゾビトキシンは、ダイズ根粒菌Bradyrhizobium elkanii USDA94株など一部の根粒菌が生産する植物のエチレン生成を抑制する物質である。より具体的には、植物のもつエチレン生合成経路を触媒する酵素の中でACC合成酵素の活性を抑制する物質である。一般に、ACC合成酵素は、植物のエチレン生合成の律速酵素であり、この酵素の活性を抑制することにより、植物のエチレン生成を抑制することができる。 Rhizobitoxin is a substance that suppresses ethylene production in plants produced by some rhizobia such as soybean rhizobia Bradyrhizobium elkanii USDA94. More specifically, it is a substance that suppresses the activity of ACC synthase among enzymes that catalyze the ethylene biosynthesis pathway of plants. In general, ACC synthase is a rate-limiting enzyme for plant ethylene biosynthesis, and by suppressing the activity of this enzyme, plant ethylene production can be suppressed.
本発明に使用したACCデアミナーゼ遺伝子は、例えば、配列番号１に示された塩基配列の情報を元にプライマーを調製し、M. loti由来のゲノムDNAを鋳型にポリメラーゼ連鎖反応（PCR）を行なうことにより増幅し、調製することができる（実施例２）。調製したACCデアミナーゼ遺伝子は、公知の大腸菌発現ベクター、例えばpUC19に挿入して、大腸菌、例えばBL21に導入して、タンパク質として発現させることができる。タンパク質は、公知の方法（Honmaら, 1978）で活性測定が可能で、調整した遺伝子が活性のあるACCデアミナーゼをコードしているかどうか確認できる（実施例３）。 For the ACC deaminase gene used in the present invention, for example, a primer is prepared based on the information of the base sequence shown in SEQ ID NO: 1, and a polymerase chain reaction (PCR) is performed using genomic DNA derived from M. loti as a template. And can be prepared (Example 2). The prepared ACC deaminase gene can be inserted into a known E. coli expression vector such as pUC19, introduced into E. coli such as BL21, and expressed as a protein. The activity of the protein can be measured by a known method (Honma et al., 1978), and it can be confirmed whether the adjusted gene encodes an active ACC deaminase (Example 3).
また、本遺伝子の一部に少数の塩基を改変した場合であっても、当該活性が維持されうることは周知の事実であり、このような改変は、当業者であれば、例えば、部分特異的変異導入法（Ausubelら, 1999, Currennt Protocols in Molecular Biology）など周知の技術を利用して行なうことができる。従って、本発明に使用するACCデアミナーゼ遺伝子は、ACCデアミナーゼ活性を有するタンパク質をコードする限り、配列表の配列番号１に記載の塩基配列において1若しくは複数の塩基が置換、欠失、付加および／または挿入などにより改変された塩基配列からなるDNAをも含むものである。ここで複数の塩基が改変された塩基配列とは、１０個以内、好ましくは５個以内、更に好ましくは３個以内の塩基が改変された塩基配列を意味するものである。 In addition, it is a well-known fact that the activity can be maintained even if a small number of bases are modified in a part of this gene. It can be carried out by using a well-known technique such as an automatic mutagenesis method (Ausubel et al., 1999, Currennt Protocols in Molecular Biology). Therefore, as long as the ACC deaminase gene used in the present invention encodes a protein having ACC deaminase activity, one or a plurality of bases in the base sequence described in SEQ ID NO: 1 in the sequence listing are substituted, deleted, added and / or It also includes DNA having a base sequence modified by insertion or the like. Here, the base sequence in which a plurality of bases are modified means a base sequence in which 10 or less, preferably 5 or less, more preferably 3 or less bases are modified.
アグロバクテリウム菌で外来遺伝子を発現させることが可能なベクターを選択し、大腸菌の発現系を使って活性を確認したACCデアミナーゼ遺伝子をそのベクターに挿入し（実施例４）、公知の方法でアグロバクテリウム菌に導入することができる（実施例５）。ACCデアミナーゼ遺伝子を保持したアグロバクテリウム菌のACCデアミナーゼ活性は、下記に述べる実施例３の方法に従って測定できる。その結果、本研究では、ACCデアミナーゼ活性を有するアグロバクテリウム菌（Agrobacterium tumefaciens C58C1RifR）を作製することができた。 A vector capable of expressing a foreign gene in Agrobacterium is selected, and an ACC deaminase gene whose activity has been confirmed using an E. coli expression system is inserted into the vector (Example 4). It can be introduced into bacteria (Example 5). The ACC deaminase activity of Agrobacterium carrying the ACC deaminase gene can be measured according to the method of Example 3 described below. As a result, in this study, Agrobacterium tumefaciens C58C1RifR having ACC deaminase activity could be produced.
さらに、ダイズ根粒菌Bradyrhizobium elkanii USDA94株のリゾビトキシンオペロンを制限酵素にて切り出し、広宿主域ベクターpBBR1MCS-2 (Kovach et al. 1995)へ、遺伝子がlacプロモーター転写制御下にあるように導入した。得られたプラスミドpBBR::PlacRTをA. tumefaciens C58C1RifR株へエレクトロポレーション法にて導入し、抗生物質カナマイシンにて導入された菌株を選択した。獲得したアグロバクテリウム菌（A. tumefaciens C58C1RifR (pBBR::PlacRT)）のリゾビトキシン生産能力をLC/MS解析により分析した結果、リゾビトキシン特有のスペクトルが検出された。したがって、A. tumefaciens C58C1RifR (pBBR::PlacRT)はリゾビトキシンを生産し、アグロバクテリウム菌への生産能付与に成功したことが明らかとなった（実施例６）。 In addition, the lysobitoxin operon of the soybean rhizobia Bradyrhizobium elkanii USDA94 was excised with restriction enzymes and introduced into the broad host range vector pBBR1MCS-2 (Kovach et al. 1995) so that the gene was under lac promoter transcriptional control. . The obtained plasmid pBBR :: PlacRT was introduced into the A. tumefaciens C58C1RifR strain by electroporation, and a strain introduced with the antibiotic kanamycin was selected. As a result of analyzing the ability of Agrobacterium (A. tumefaciens C58C1RifR (pBBR :: PlacRT)) to produce lysobitoxin by LC / MS analysis, a spectrum specific to lysobitoxin was detected. Therefore, it was revealed that A. tumefaciens C58C1RifR (pBBR :: PlacRT) produced lysobitoxin and succeeded in conferring production ability to Agrobacterium (Example 6).
下記の実施例において、コスミドクローン（ACCESSION AB 062279）のrxtAからオープンリーディングフレーム４（ORF4）に当たる約9kbのリゾビトキシンオペロン配列を切り出して使用した。その約9kbに相当する部分の塩基配列を、配列表の配列番号２に示す。本発明に使用するリゾビトキシンオペロンは、リゾビトキシン生産能力を付与することができる限り、配列番号２に記載の塩基配列において1若しくは複数の塩基が置換、欠失、付加および／または挿入などにより改変された塩基配列からなるDNAをも含むものである。ここで複数の塩基が改変された塩基配列とは、１０個以内、好ましくは５個以内、更に好ましくは３個以内の塩基が改変された塩基配列を意味するものである。 In the following examples, an approximately 9 kb lysobitoxin operon sequence corresponding to the open reading frame 4 (ORF4) was cut out from rxtA of the cosmid clone (ACCESSION AB 062279) and used. The base sequence corresponding to about 9 kb is shown in SEQ ID NO: 2 in the sequence listing. The lysovitoxin operon used in the present invention is modified by substitution, deletion, addition and / or insertion of one or more bases in the base sequence described in SEQ ID NO: 2 as long as it can confer lysovitoxin production ability It also includes DNA consisting of the determined base sequence. Here, the base sequence in which a plurality of bases are modified means a base sequence in which 10 or less, preferably 5 or less, more preferably 3 or less bases are modified.
なお本発明におけるリゾビトキシン生合成系の遺伝子とは、遺伝子が導入された宿主にリゾビトキシンを生合成する能力を付与することができる種々の遺伝子を意味するものである。実施例６ではリゾビトキシンオペロンを採用しているが、それに限定されるものではなく、リゾビトキシンを生合成する能力を付与する能力を有する限り、他のリゾビトキシン生合成系の遺伝子を使用することも本発明の範囲内である。 In addition, the gene of the lysobitoxin biosynthesis system in the present invention means various genes that can confer the ability to biosynthesize lysobitoxin to the host into which the gene has been introduced. Although the lysobitoxin operon is employed in Example 6, the present invention is not limited to this, and other lysobitoxin biosynthetic genes may be used as long as they have the ability to impart the ability to biosynthesize lysobitoxin. It is within the scope of the present invention.
更に、ACCデアミナーゼ活性を付与したアグロバクテリウム菌を使用して、植物への遺伝子導入能の評価を行った。その結果、ACCデアミナーゼ活性を付与したアグロバクテリウム菌（A. tumefaciens C58C1RifR）では、指標となるＧＵＳ遺伝子の発現が増加しており、植物への外来遺伝子導入効率が向上していることが示された（実施例７）。 Furthermore, the ability of gene introduction into plants was evaluated using Agrobacterium given ACC deaminase activity. As a result, in Agrobacterium (A. tumefaciens C58C1RifR) imparted with ACC deaminase activity, the expression of the GUS gene serving as an index is increased, indicating that the efficiency of introducing a foreign gene into a plant is improved. (Example 7).
植物のエチレン生成を抑制する物質を生産する遺伝子をアグロバクテリウム菌中で発現させる方法はベクターを用いた方法に制限されない。例えば、アグロバクテリウム菌のゲノムDNA中に挿入して発現することもできる。 A method of expressing a gene that produces a substance that suppresses plant ethylene production in Agrobacterium is not limited to a method using a vector. For example, it can be expressed by inserting into the genomic DNA of Agrobacterium.
上記本発明で作製したアグロバクテリウム菌を用いて植物に遺伝子を導入する場合、導入する外来遺伝子を植物発現用に開発された公知のベクターに挿入し、アグロバクテリウム菌に導入する。例えば、汎用されているpBI121やpBI221などの植物発現用に開発されたベクターのマーカー遺伝子部分を制限酵素を用いて切り出し、これに代えて目的外来遺伝子を導入する。構築したベクターは、公知の方法によりアグロバクテリウム菌に導入することができる。 When a gene is introduced into a plant using the Agrobacterium produced in the present invention, the introduced foreign gene is inserted into a known vector developed for plant expression and introduced into Agrobacterium. For example, a marker gene portion of a vector developed for plant expression such as pBI121 or pBI221 which is widely used is cut out using a restriction enzyme, and a target foreign gene is introduced instead. The constructed vector can be introduced into Agrobacterium by a known method.
以上により作製したアグロバクテリウム菌を公知のアグロバクテリウムによる遺伝子導入法に利用することにより、植物細胞への効率的な遺伝子導入を行うことができる。アグロバクテリウム法では、pBI121由来のベクターを好適に用いることができ、ベクターを導入する植物細胞の形態としては、組織・器官・カルスの何れの形態でもよい。本研究で作製したアグロバクテリウムベクターが遺伝子導入をする植物細胞の種類としては、特に制限はなく、双子葉植物であっても単子葉植物であってもよい。遺伝子導入効率の高低はあるものの、アグロバクテリム法による遺伝子導入の報告が、イネ、トマト、メロンなど広く植物において用いられている。 By using the Agrobacterium prepared as described above for a known Agrobacterium gene transfer method, efficient gene transfer into plant cells can be performed. In the Agrobacterium method, a pBI121-derived vector can be suitably used, and the form of the plant cell into which the vector is introduced may be any form of tissue, organ, and callus. There are no particular restrictions on the types of plant cells into which the Agrobacterium vector produced in this study introduces genes, and it may be a dicotyledonous plant or a monocotyledonous plant. Although there are high and low gene transfer efficiency, reports of gene transfer by the Agrobacterium method are widely used in plants such as rice, tomato, and melon.
アグロバクテリウム法によりベクターを導入した植物細胞は、一般に、形質転換細胞を選抜する抗生物質とアグロバクテリウム菌を除菌するための抗生物質、及び植物体再生用の植物ホルモン（オーキシン、サイトカイニンなど）を含む組織培養用の培地で培養し、植物体の再生を行うことができる。エレクトロポレーション法及びパーティクルガン法でベクターを導入した植物細胞は、一般に、形質転換細胞を選抜する抗生物質と植物体再生用の植物ホルモン（オーキシン、サイトカイニンなど）を含む組織培養用の培地で培養し、植物体の再生を行うことができる。 Plant cells into which a vector has been introduced by the Agrobacterium method are generally divided into antibiotics for selecting transformed cells, antibiotics for eliminating Agrobacterium, and plant hormones for plant regeneration (auxin, cytokinin, etc.) The plant body can be regenerated by culturing in a tissue culture medium containing Plant cells into which vectors have been introduced by the electroporation method and particle gun method are generally cultured in a tissue culture medium containing antibiotics for selecting transformed cells and plant hormones for plant regeneration (auxin, cytokinin, etc.). Then, the plant body can be regenerated.
例えば、メロンでは以下の方法を用いることができる。メロンの種子を濾紙上に無菌播種し、２５℃、１６時間照明（約4,000lux）で発芽させる。５日〜７日後に展開した子葉を６分割し、導入したい遺伝子を持つアグロバクテリウム菌（A. tumefaciens LBA4404, C58C1RifR株など）を接種する。BAP1mg/lを含むMS培地上で３日間共存培養する。続いて、BAP1mg/l, クラフォラン500mg/lを含むMS培地上で１週間除菌のための培養を行なう。更に、BAP1mg/l, クラフォラン500mg/l, カナマイシン 200mg/lを含むMS培地上で2週間毎に継代培養する。伸長してきたシュートをクラフォラン500mg/l, カナマイシン 100mg/lを含むMS培地に継代して発根させる。得られた再生個体について遺伝子の導入の確認を行ない、形質転換体が得られた事を確認する。
For example, the following method can be used for melon. Melon seeds are aseptically sown on filter paper and germinated at 25 ° C. for 16 hours with illumination (about 4,000 lux). 5 to 7 days later, the expanded cotyledons are divided into 6 and inoculated with Agrobacterium (A. tumefaciens LBA4404, C58C1RifR, etc.) having the gene to be introduced. Co-culture for 3 days on MS medium containing 1 mg / l BAP. Subsequently, culture for sterilization is performed for 1 week on an MS medium containing 1 mg / l of BAP and 500 mg / l of claforan. Furthermore, the cells are subcultured every 2 weeks on MS medium containing 1 mg / l of BAP, 500 mg / l of claforan and 200 mg / l of kanamycin. Elongate shoots are rooted by subculture in MS medium containing kraforan 500 mg / l,
また、トマトでは、例えば、文献（Ohyamaら, 1995, Plant Cell Physiology, 36:369-376）に記載の方法を用いることができる。具体的には、トマトの種子をMS培地上に無菌播種し、２５℃、１６時間照明（約4,000lux）で発芽させる。発芽してきた実生の子葉を２分割し、導入したい遺伝子を持つアグロバクテリウム菌（A. tumefaciens LBA4404, C58C1RifR株など)を接種する。ゼアチン1mg/lを含むMS培地上で2日間共存培養する。続いて、ゼアチン1mg/l, カルベニシリン 200mg/lを含むMS培地上で１週間除菌のための培養を行う。更に、Zeatin1mg/l, カルベニシリン200mg/l, カナマイシン 100mg/lを含むMS培地上で2週間毎に継代培養する。伸長してきたシュートをカルベニシリン200mg/l, カナマイシン 50 mg/lを含むMS培地に継代して発根させる。得られた再生個体について、導入遺伝子の確認を行い形質転換体を確認する。 For tomato, for example, the method described in the literature (Ohyama et al., 1995, Plant Cell Physiology, 36: 369-376) can be used. Specifically, tomato seeds are aseptically sown on MS medium and germinated with illumination (about 4,000 lux) at 25 ° C. for 16 hours. Divided seedling cotyledons that have germinated are inoculated with Agrobacterium (A. tumefaciens LBA4404, C58C1RifR, etc.) having the gene to be introduced. Co-culture for 2 days on MS medium containing 1 mg / l zeatin. Subsequently, culture for sterilization is performed for 1 week on an MS medium containing 1 mg / l zeatin and 200 mg / l carbenicillin. Furthermore, the cells are subcultured every 2 weeks on MS medium containing 1 mg / l of Zeatin, 200 mg / l of carbenicillin and 100 mg / l of kanamycin. Elongate shoots are rooted by passage into MS medium containing 200 mg / l carbenicillin and 50 mg / l kanamycin. For the obtained regenerated individuals, the transgene is confirmed and the transformant is confirmed.
これにより形質転換植物体が作出されれば、その繁殖材料（例えば、果実、種子など）を得ることができる。さらに、得られた繁殖材料を基に形質転換植物体を量産することも可能である。 Thus, if a transformed plant body is produced, its propagation material (for example, fruit, seed, etc.) can be obtained. Furthermore, it is possible to mass-produce transformed plants based on the obtained propagation material.
以下、本発明を実施例によりさらに詳細に説明するが、本発明はこれら実施例に制限されるものではない。 EXAMPLES Hereinafter, although an Example demonstrates this invention further in detail, this invention is not restrict | limited to these Examples.
実施例１：アグロバクテリウム法による植物への遺伝子導入に対するエチレンの影響
本実験では、アグロバクテリウムAgrobacterium tumefaciens 菌株C58C1RifR(Van Larebeke et al., 1974)を用いた。遺伝子導入ヘルパープラスミドとしてpIG121-Hm (Akama et al. 1992)を用いた。このプラスミドの T-DNA にある遺伝子 uidA に担われた酵素β-グルクロニダーゼ活性を本研究での遺伝子導入の指標として用いた。本遺伝子は pIG121-Hm 上のT-DNA 領域において、カリフラワーモザイクウイルス由来35Sプロモーター及び本遺伝子の内部に挿入されたヒマ由来(castor bean、Ricinus communis L.)カタラーゼ遺伝子のイントロン配列・ノパリン合成酵素遺伝子の転写終結配列に結合されている。このプラスミド pIG121-Hmをエレクトロポレーション法(Formm et al., 1985)によりA. tumefaciens C58C1RifRへ形質転換した(以降A. tumafaciens C58C1RifR (pIG121-Hm)と呼ぶ) 。この形質転換菌株は、抗生物質 カナマイシン (100mg/L) と テトラサイクリン (1mg/L) を含む LB 培地で選抜して維持した。
Example 1: Effect of ethylene on gene introduction into plants by Agrobacterium method In this experiment, Agrobacterium Agrobacterium tumefaciens strain C58C1RifR (Van Larebeke et al., 1974) was used. PIG121-Hm (Akama et al. 1992) was used as a gene transfer helper plasmid. The enzyme β-glucuronidase activity carried by the gene uidA in the T-DNA of this plasmid was used as an index for gene transfer in this study. In the T-DNA region on pIG121-Hm, this gene is a 35S promoter derived from cauliflower mosaic virus and an intron sequence of castor bean, Ricinus communis L. catalase gene inserted into this gene, nopaline synthase gene To the transcription termination sequence. This plasmid pIG121-Hm was transformed into A. tumefaciens C58C1RifR by electroporation (Formm et al., 1985) (hereinafter referred to as A. tumafaciens C58C1RifR (pIG121-Hm)). This transformed strain was selected and maintained in LB medium containing the antibiotics kanamycin (100 mg / L) and tetracycline (1 mg / L).
植物への遺伝子導入に対するエチレンの影響を評価するために、エチレン前駆物質 ACC を添加して、A.tumefaciens C58C1RifR (pIG121-Hm)を接種したメロン子葉外植片を共存培地上で培養した。図１において、A. tumefaciens C58C1RifR(pIG121-Hm)(Akama et al., 1992)を接種したメロン子葉外植片において、β-グルクロニダーゼ酵素活性を測定した結果を示す。なお接種は４日間行なった。図１において１本のカラムは、外植片１枚から抽出された全蛋白質量当たりの本酵素の活性を示している。また黒と白のカラムはそれぞれ、200 uM・ACC 添加、ACC無添加を示している。無添加区（白カラム）におけるβ-グルクロニダーゼ酵素活性は、培養期間を通じて経日的に上昇した。200 uM・ACC添加区（黒カラム）における本酵素の活性は、接種後２日目から認められたが、３日目以後はほとんど増加しなかった。 In order to evaluate the influence of ethylene on gene transfer into plants, melon cotyledon explants inoculated with A. tumefaciens C58C1RifR (pIG121-Hm) were cultured on a co-culture medium with the addition of the ethylene precursor ACC. FIG. 1 shows the results of measuring β-glucuronidase enzyme activity in melon cotyledon explants inoculated with A. tumefaciens C58C1RifR (pIG121-Hm) (Akama et al., 1992). The inoculation was performed for 4 days. In FIG. 1, one column shows the activity of this enzyme per total protein extracted from one explant. The black and white columns indicate the addition of 200 uM · ACC and no ACC, respectively. The β-glucuronidase enzyme activity in the non-added section (white column) increased with time throughout the culture period. The activity of this enzyme in the 200 uM · ACC addition group (black column) was observed from the second day after the inoculation, but hardly increased after the third day.
また、A. tumefaciens C58C1RifR(pIG121-Hm)を接種したメロン子葉外植片において、エチレン発生速度を測定した（図２）。なお接種は４日間行なった。図２において各点は外植片のエチレン発生速度を示している。図２において、黒丸と白丸はそれぞれ、200 uM・ACC 添加区とACC無添加を示す。黒三角はアグロバクテリウム菌無接種区を示している。全培養期間を通じて、200 uM・ACC 添加区におけるエチレン発生速度は、無添加区の約100 倍となっていた。これらの結果は、エチレン発生の増加が、植物への遺伝子導入を抑制することを示す。 In addition, ethylene generation rates were measured in melon cotyledon explants inoculated with A. tumefaciens C58C1RifR (pIG121-Hm) (FIG. 2). The inoculation was performed for 4 days. In FIG. 2, each point indicates the ethylene generation rate of the explant. In FIG. 2, the black circles and white circles indicate the 200 uM · ACC added group and the non-ACC added group, respectively. The black triangle indicates the non-inoculated area of Agrobacterium. Throughout the entire culture period, the ethylene generation rate in the 200 uM / ACC-added group was about 100 times that in the non-added group. These results indicate that increased ethylene generation suppresses gene transfer into plants.
続いて、内生エチレンの影響を評価するために、エチレン生合成阻害剤AVG を添加して、A. tumefaciens C58C1RifR (pIG121-Hm)を接種したメロン子葉外植片を共存培地上で培養した（図３）。A. tumefaciens C58C1RifR (pIG121-Hm)を接種したメロン子葉外植片において、β-グルクロニダーゼ酵素活性を測定した。なお共存培養は４日間行った。１本のカラムは、外植片１枚から抽出された全蛋白質量当たりの本酵素の活性を示している。白と黒カラムはそれぞれ、AVG 無添加区と10 uM ・ AVG 添加区を示している。AVG無添加区（白カラム）におけるβ-グルクロニダーゼ酵素活性は、培養期間を通じて経日的に上昇した。一方10 uM・AVG 添加区（黒カラム）における本酵素の活性は、無添加区と比べて著しく高くなった。 Subsequently, in order to evaluate the effects of endogenous ethylene, melon cotyledon explants inoculated with A. tumefaciens C58C1RifR (pIG121-Hm) were cultured on a co-culture medium with the addition of the ethylene biosynthesis inhibitor AVG ( FIG. 3). Β-glucuronidase enzyme activity was measured in melon cotyledon explants inoculated with A. tumefaciens C58C1RifR (pIG121-Hm). The coculture was performed for 4 days. One column shows the activity of the enzyme per total protein extracted from one explant. The white and black columns indicate the AVG-free group and the 10 uM AVG-added group, respectively. The β-glucuronidase enzyme activity in the AVG-free group (white column) increased with time throughout the culture period. On the other hand, the activity of this enzyme in the 10 uM / AVG added group (black column) was significantly higher than that in the non-added group.
A. tumefaciens C58C1RifR (pIG121-Hm) を接種したメロン子葉外植片において、エチレン発生速度を測定した（図４）。なお接種は４日間行なった。１つの点は外植片のエチレン発生速度を示している。白丸と黒丸はそれぞれ、AVG 無添加区、10 uM ・AVG 添加区を示している。また黒三角はアグロバクテリウム菌無接種区を示している。培養期間を通じて、10 uM・AVG 添加区（黒丸）において、エチレンの発生はほとんど検出されなかった。エチレン発生が阻害された条件での遺伝子導入の促進は、内生エチレンが植物への遺伝子導入を抑制していることを示す。これらの事実は、内生エチレン発生の阻害が、遺伝子導入の効率を向上させる改良方法であることを示す。 The rate of ethylene generation was measured in melon cotyledon explants inoculated with A. tumefaciens C58C1RifR (pIG121-Hm) (FIG. 4). The inoculation was performed for 4 days. One point shows the ethylene generation rate of the explant. The white and black circles indicate the AVG-free group and the 10 uM / AVG-added group, respectively. The black triangles indicate the non-inoculated area of Agrobacterium. Throughout the culture period, almost no ethylene was detected in the 10 uM · AVG addition group (black circle). The promotion of gene transfer under conditions where ethylene generation is inhibited indicates that endogenous ethylene suppresses gene transfer into plants. These facts indicate that inhibition of endogenous ethylene generation is an improved method for improving the efficiency of gene transfer.
また図５において、メロン子葉外植片における A. tumefaciens C58C1RifR (pIG121-Hm)の生育に対する、エチレンの影響を調査した。接種4 日後の外植片の洗浄水を培地に塗布し、形成されたコロニーを計数した。その結果、ACC 添加区（黒カラム）とACC無添加区（白カラム）において有意な差は認められず、A. tumefaciens C58C1RifR (pIG121-Hm)の生育に対してエチレンは影響を及ぼさないと考えられた。 Further, in FIG. 5, the influence of ethylene on the growth of A. tumefaciens C58C1RifR (pIG121-Hm) in melon cotyledon explants was investigated. Four days after inoculation, the explant washing water was applied to the medium, and the formed colonies were counted. As a result, there was no significant difference between the ACC-added group (black column) and the ACC-free group (white column), and ethylene was considered to have no effect on the growth of A. tumefaciens C58C1RifR (pIG121-Hm). It was.
なお実施例１において、A. tumafaciens C58C1RifR(pIG121-Hm)は、36時間、28℃で振盪培養した。集められた菌体細胞は、MS培地で洗浄したあと、MS培地に懸濁した。この細胞懸濁液は、O.D.= 0.5(600 nm)になるよう希釈して、接種実験に用いた(107 cells/mL)。メロン子葉の無菌外植片は、 C58C1RifR (pIG121-Hm)の細胞懸濁液に20分間浸したあと、MS培地で洗浄した。洗浄した外植片は、MS 培地に置き、25℃、暗黒下で培養した。エチレン前駆物質ACC(1-アミノシクロプロパン-1-カルボン酸)またはエチレン生合成阻害剤AVG (アミノエトキシビニルグリシン)の投与は、MS培地にあらかじめ添加することにより行なった。これらエチレン関連化合物によるエチレン発生の影響は、ガスクロマトグラフィー (Shimadzu, Tokyo, Japan)を用いて評価した(検出限界 0.1 uL/L)。 In Example 1, A. tumafaciens C58C1RifR (pIG121-Hm) was cultured with shaking at 28 ° C. for 36 hours. The collected bacterial cells were washed with MS medium and then suspended in MS medium. This cell suspension was diluted to O.D. = 0.5 (600 nm) and used for inoculation experiments (107 cells / mL). The melon cotyledon explant was immersed in a cell suspension of C58C1RifR (pIG121-Hm) for 20 minutes and then washed with MS medium. The washed explants were placed in MS medium and cultured at 25 ° C. in the dark. Administration of the ethylene precursor ACC (1-aminocyclopropane-1-carboxylic acid) or the ethylene biosynthesis inhibitor AVG (aminoethoxyvinylglycine) was performed by pre-adding to the MS medium. The effects of ethylene generation by these ethylene-related compounds were evaluated using gas chromatography (Shimadzu, Tokyo, Japan) (detection limit 0.1 uL / L).
外植片を接種する植物材料としてはメロン品種ベドランテス(Cucumis melo L. var. cantalopensis cv. Vedrantais)の種子を用いた。ベドランテスの種子は70%エタノールに10 秒間浸したあと、2%次亜塩素酸ナトリウム溶液、0.02% Tween 20溶液に30分間浸して表面殺菌を行い、1/2濃度のMS培地(Murashige et al., 1962)に播種した。播種した種子を25℃、50μE/m2/s、16時間の日長で生育させた。播種後６日目の子葉は、コルクボーラでくりぬき、無菌外植片として接種実験に用いた。 As plant material to inoculate explants, seeds of melon cultivar Vedrantes (Cucumis melo L. var. Cantalopensis cv. Vedrantais) were used. Vedrantes seeds were soaked in 70% ethanol for 10 seconds, then immersed in 2% sodium hypochlorite solution and 0.02% Tween 20 solution for 30 minutes for surface sterilization, and ½ concentration MS medium (Murashige et al. , 1962). The sown seeds were grown at 25 ° C., 50 μE / m 2 / s, with a day length of 16 hours. The cotyledon 6 days after sowing was hollowed out with a cork borer and used as a sterile explant for inoculation experiments.
外植片への遺伝子導入の評価は、T-DNA を持つ外植片の粗酵素抽出液に認められるβ-グルクロニダーゼ酵素活性を利用した。外植片は、タンパク質抽出バッファー(50 mM・ナトリウムリン酸バッファー-10 mM・EDTA-0.1%・TritonX-100-0.1%・N-ドデカノイルサルコシン酸ナトリウム-10 mM・2-メルカプトエタノール、pH 7.0)に漬けて、氷冷しながら破砕した。この破砕液は遠心分離して、その上清を得た。 The evaluation of gene transfer into explants used β-glucuronidase enzyme activity found in the crude enzyme extract of explants with T-DNA. The explants consisted of protein extraction buffer (50 mM, sodium phosphate buffer-10 mM, EDTA-0.1%, TritonX-100-0.1%, sodium N-dodecanoyl sarcosate-10 mM, 2-mercaptoethanol, pH 7.0 ) And crushed with ice cooling. The crushed liquid was centrifuged to obtain the supernatant.
この上清に対して 2.2 倍量のタンパク質抽出バッファを混合させて、外植片の粗酵素抽出液とした。粗酵素抽出液のタンパク質濃度は、Bio-Rad Protein Assay Dye Reagent Concentrate(BIO RAD, Tokyo)を用いて、あらかじめ測定した。酵素β-グルクロニダーゼは、基質4-MUG(4-メチル-ウンベリルフェリル-β-D-グルクロニド)を、β-D-グルクロニドと4-MU(4-メチルウンベリルフェロン)に分解する。この分解産物4-MU は、紫外光(365 nm)により蛍光を発する。 The supernatant was mixed with 2.2 times the amount of protein extraction buffer to obtain a crude enzyme extract of explants. The protein concentration of the crude enzyme extract was measured in advance using Bio-Rad Protein Assay Dye Reagent Concentrate (BIO RAD, Tokyo). The enzyme β-glucuronidase degrades the substrate 4-MUG (4-methyl-umbellyl ferryl-β-D-glucuronide) into β-D-glucuronide and 4-MU (4-methylumbellyl ferron). This degradation product 4-MU emits fluorescence by ultraviolet light (365 nm).
粗酵素抽出液と同量の基質液(1 mM・4-MUG-50mM・ナトリウムリン酸バッファー-10 mM・EDTA-0.1%・TritonX-100-0.1%・N-ドデカノイルサルコシン酸ナトリウム-10 mM・2-メルカプトエタノール、pH7.0)を加えて、37℃で保温して反応させた。反応を停止させるために、反応液の20倍量の停止液(0.2 M・Na2CO3)を加えた。反応を止めた試料の持つ、紫外光595nmにおける蛍光強度は、分光蛍光光度計で測定した。 Substrate solution (1 mM, 4-MUG-50 mM, sodium phosphate buffer-10 mM, EDTA-0.1%, TritonX-100-0.1%, sodium N-dodecanoyl sarcosate-10 mM -2-Mercaptoethanol, pH 7.0) was added, and the mixture was incubated at 37 ° C for reaction. In order to stop the reaction, 20 times the amount of the stop solution (0.2 M · Na 2 CO 3 ) was added. The fluorescence intensity at 595 nm of ultraviolet light possessed by the sample that stopped the reaction was measured with a spectrofluorometer.
反応停止液に混合した反応産物4-MUの希釈系列(2 nMから1000 nM)を用いて、反応停止液の蛍光強度と4-MU濃度を対応させた。粗酵素抽出液のβ-グルクロニダーゼ酵素活性(時間当たり・タンパク質重量当たりの4-MU分子の蓄積量)を植物への遺伝子導入の指標とした。 Using a dilution series (2 nM to 1000 nM) of the reaction product 4-MU mixed with the reaction stop solution, the fluorescence intensity of the reaction stop solution was associated with the 4-MU concentration. The β-glucuronidase enzyme activity (accumulated amount of 4-MU molecules per hour / protein weight) of the crude enzyme extract was used as an index for gene introduction into plants.
実施例２：ACCデアミナーゼ遺伝子のクローニングとシークエンス解析
本実験では、ACCデアミナーゼ遺伝子をクローニングする材料としてミヤコグサ根粒菌 Mesorhizobium loti 菌株 MAFF 303099を用い、以下の方法により遺伝子を獲得した。実験に際して本菌を28 ℃、HM 培地(Cole et al., 1973) で、生育・維持した。M. loti MAFF303099のトータルDNAをSambrook et al.（2001）の方法により抽出して、PCR の鋳型とした。本株のゲノム情報(Kaneko et al., 2000)に基づいてACC デアミナーゼ様遺伝子mlr5932のクローンを得るために、２つのプライマーacdsF(5’- TGCGGATCCT GACTAAGGAG GAGAGAGAAT ATGCTGGAAA AAATCCAGC-3’)およびacdsR(5’-TGCGGTACCG TTGATCAGCC ATTGCGAAAG GTGAGCC-3’)を設計した。
Example 2: Cloning and sequence analysis of ACC deaminase gene In the present experiment, the acc deaminase gene was used as a material for cloning the ACC deaminase gene, and the gene was obtained by the following method using Mesorhizobium loti strain MAFF 303099. During the experiment, this bacterium was grown and maintained in HM medium (Cole et al., 1973) at 28 ° C. The total DNA of M. loti MAFF303099 was extracted by the method of Sambrook et al. (2001) and used as a PCR template. To obtain a clone of the ACC deaminase-like gene mlr5932 based on the genome information of this strain (Kaneko et al., 2000), two primers acdsF (5'-TGCGGATCCT GACTAAGGAG GAGAGAGAAT ATGCTGGAAA AAATCCAGC-3 ') and acdsR (5' -TGCGGTACCG TTGATCAGCC ATTGCGAAAG GTGAGCC-3 ') was designed.
抽出したトータルDNAと設計したプライマーを用いて、95℃、9分間の熱変性のあと、94℃で1分間、66℃で1分間、72℃で1分間、この温度サイクルを40回繰り返すという条件で PCR を行った。期待した大きさ1.0kbのPCR産物は、クローニングベクターpGEM-T(Promega, USA)にサブクローニングした。この増幅断片は BamHIと KpnIで切断し、lacプロモーターの転写制御下になるようpUC19にサブクローニングした(pUC19acds)。クローン化した断片は、DNAシークエンサーABI310とDNA Sequencing Kit Big Dye Terminator cycle sequencing Ready Reaction(Applied Biosystems, Tokyo)を用いて、塩基配列の決定を行い、データベース上に登録した配列(AP003007:mlr5932)と同一であることを確認した。 Using the extracted total DNA and the designed primers, heat denaturation at 95 ° C for 9 minutes, then 94 ° C for 1 minute, 66 ° C for 1 minute, 72 ° C for 1 minute, 40 cycles of this temperature cycle PCR was carried out. The expected 1.0 kb PCR product was subcloned into the cloning vector pGEM-T (Promega, USA). This amplified fragment was cleaved with BamHI and KpnI and subcloned into pUC19 under the transcriptional control of the lac promoter (pUC19acds). The cloned fragment was determined using the DNA sequencer ABI310 and DNA Sequencing Kit Big Dye Terminator cycle sequencing Ready Reaction (Applied Biosystems, Tokyo), and the same sequence as that registered on the database (AP003007: mlr5932) It was confirmed that.
図６に、M loti 由来ACCデアミナーゼ遺伝子 mlr5932 とPseudomonas 由来ACCデアミナーゼ遺伝子の塩基配列を比較した図を示す。２つの遺伝子、即ちmlr5932 (上段：Genbank accession number AP003007)とM73488（下段）の塩基配列の間には、68.1%の相同性が認められた。 FIG. 6 shows a comparison of the base sequences of the M loti-derived ACC deaminase gene mlr5932 and the Pseudomonas-derived ACC deaminase gene. 68.1% homology was observed between the base sequences of two genes, mlr5932 (top: Genbank accession number AP003007) and M73488 (bottom).
更に図７に、M loti 由来遺伝子ACCデアミナーゼ遺伝子 mlr5932 の推定アミノ酸コード領域と Pseudomonas 由来 ACC デアミナーゼのアミノ酸配列を比較した図を示す。２つの遺伝子の推定アミノ酸配列、即ちmlr5932 (上段：Genbank accession number BAB52295.1)とAAA25689.1（下段）のアミノ酸配列の間には、69.3%の相同性が認められた。 Further, FIG. 7 shows a diagram comparing the deduced amino acid coding region of M loti-derived gene ACC deaminase gene mlr5932 and the amino acid sequence of Pseudomonas-derived ACC deaminase. 69.3% homology was observed between the deduced amino acid sequences of the two genes, namely mlr5932 (upper: Genbank accession number BAB52295.1) and AAA25689.1 (lower).
実施例３：ACCデアミナーゼ遺伝子の機能解析
実施例２で作製したクローン mlr5932 を保持する大腸菌BL21(pUC19acds)およびBL21 (pUC19)の粗酵素抽出液のACC 添加後のα-ケト酪酸の蓄積速度を観察した。大腸菌 E. coli BL21(pUC19acds)、および対照としたBL21(pUC19)の粗酵素抽出液において、ACC 添加後のα-ケト酪酸の蓄積速度を測定した結果を図８に示す。図８においてカラムとエラーバーはそれぞれ、平均値と標準偏差を示している。IPTG の添加は、BL21(pUC19acds)、BL21(pUC19)の粗酵素抽出液におけるα-ケト酪酸の蓄積速度を上昇させた。このとき、BL21(pUC19acds)におけるα-ケト酪酸の蓄積速度（黒カラム）は、BL21(pUC19) におけるα-ケト酪酸の蓄積速度（白カラム）に比べて明らかに大きくなった。
Example 3: Functional analysis of ACC deaminase gene Observation of accumulation rate of α-ketobutyric acid after addition of ACC in crude enzyme extract of E. coli BL21 (pUC19acds) and BL21 (pUC19) carrying clone mlr5932 prepared in Example 2 did. FIG. 8 shows the results of measuring the accumulation rate of α-ketobutyric acid after addition of ACC in the crude enzyme extract of E. coli BL21 (pUC19acds) and BL21 (pUC19) as a control. In FIG. 8, the column and the error bar indicate the average value and the standard deviation, respectively. The addition of IPTG increased the accumulation rate of α-ketobutyric acid in the crude enzyme extract of BL21 (pUC19acds) and BL21 (pUC19). At this time, the accumulation rate of α-ketobutyric acid in BL21 (pUC19acds) (black column) was clearly larger than the accumulation rate of α-ketobutyric acid in BL21 (pUC19) (white column).
プラスミドpUC19acdsにおけるクローンmlr5932の転写はlacプロモーターの制御下にあり、誘導物質IPTGにより転写が促進されると期待できる。すなわち、IPTG の添加はmlr5932の転写を促進し、mlr5932の翻訳産物がα-ケト酪酸の蓄積速度の増大に寄与したと解釈できる。遺伝子mlr5932が既知のACCデアミナーゼ遺伝子と類似すること、その誘導とαケト酪酸の蓄積速度の増大が関連したことから、ミヤコグサ根粒菌由来遺伝子mlr5932が、機能のあるACCデアミナーゼをコードすると結論できた。 Transcription of clone mlr5932 in plasmid pUC19acds is under the control of the lac promoter and can be expected to be promoted by the inducer IPTG. That is, it can be interpreted that the addition of IPTG promoted the transcription of mlr5932, and that the translation product of mlr5932 contributed to the increase in the accumulation rate of α-ketobutyric acid. Since the gene mlr5932 is similar to the known ACC deaminase gene, and its induction was associated with an increase in the accumulation rate of α-ketobutyric acid, it was concluded that the gene from Rhizobium rhizobia encodes a functional ACC deaminase.
実施例３において、ACC デアミナーゼ活性はHonma et al.(1978)の方法を改良して測定した。集菌した細菌細胞は、0.1 M カリウムリン酸バッファー(pH7.5)で２回洗浄したあと、このバッファーに懸濁した。懸濁液を冷却しながら超音波破砕機により細胞を破砕した。細胞破砕液は遠心して、その上清を粗酵素抽出液とした。粗酵素抽出液の一部は、タンパク質濃度の定量に用いた。酵素 ACC デアミナーゼは、基質 ACC をα-ケト酪酸とアンモニアに分解する(Honma et al., 1978)。ACCデアミナーゼ酵素反応液は、粗酵素抽出液：反応バッファー(0.2 M・Tris-HCl、pH 8.5)：基質溶液(0.2 M・ACC)を、１：２：１の比で混合させて調製した。 In Example 3, ACC deaminase activity was measured by modifying the method of Honma et al. (1978). The collected bacterial cells were washed twice with 0.1 M potassium phosphate buffer (pH 7.5) and then suspended in this buffer. The cells were disrupted by an ultrasonic disrupter while the suspension was cooled. The cell lysate was centrifuged and the supernatant was used as a crude enzyme extract. A part of the crude enzyme extract was used for quantification of protein concentration. The enzyme ACC deaminase degrades the substrate ACC into α-ketobutyric acid and ammonia (Honma et al., 1978). The ACC deaminase enzyme reaction solution was prepared by mixing crude enzyme extract: reaction buffer (0.2 M · Tris-HCl, pH 8.5): substrate solution (0.2 M · ACC) at a ratio of 1: 2: 1.
この反応液を30℃で保温し、一定時間後に反応液の９倍量の停止液(0.56 N・ HCl) を加えた。この反応停止液に、0.15倍量の呈色液(1 %・2,4-デニトロフェニルヒドラジン-2N・HCl)を加えて15分間、30℃で保温した。15分間のインキュベーションの後、反応液と同量の2N・NaOHを加え呈色反応を停止した。その呈色反応液中に含まれるα-ケト酪酸は、540 nmの吸収波長で検出した(Reitman et al., 1957)。0.1 M・リン酸カリウム緩衝液(pH7.5)に溶解したα-ケト酪酸の希釈系列は、検量線の作製に使用した。酵素活性の測定実験の確認は、Pseudomonas由来のACCデアミナーゼ遺伝子を保持した大腸菌E. coli SURE株(Sheehy, et al. 1991)を利用した。この菌株は100 mg/Lのアンピシリンを含むLB培地で培養し、細胞を集めた。この細胞の破砕液は前述の測定方法に用いられ、そのACCデアミナーゼ酵素活性を検出することができた(100 nmol・mg protein-1・min-1)。 The reaction solution was kept at 30 ° C., and after a certain time, 9 times the amount of the stop solution (0.56 N · HCl) was added. To this reaction stop solution, 0.15 times the amount of the color developing solution (1% · 2,4-denitrophenylhydrazine-2N · HCl) was added and kept at 30 ° C. for 15 minutes. After 15 minutes of incubation, the same amount of 2N · NaOH as the reaction solution was added to stop the color reaction. Α-ketobutyric acid contained in the color reaction solution was detected at an absorption wavelength of 540 nm (Reitman et al., 1957). A dilution series of α-ketobutyric acid dissolved in 0.1 M potassium phosphate buffer (pH 7.5) was used to prepare a calibration curve. Confirmation of the enzyme activity measurement experiment utilized E. coli SURE strain (Sheehy, et al. 1991) carrying the ACC deaminase gene derived from Pseudomonas. This strain was cultured in LB medium containing 100 mg / L ampicillin, and the cells were collected. The cell lysate was used in the above-described measurement method, and its ACC deaminase enzyme activity could be detected (100 nmol · mg protein −1 · min −1 ).
実施例４：アグロバクテリウム発現ベクターの構築と導入
このプラスミドを保持する大腸菌E. coli BL21(pUC19acds)からプラスミドpUC19acdsを常法に従って抽出した。プラスミドベクター pUC19 に mlr5932 のアミノ酸コード領域を結合させた、Mesorhizobium loti 由来 mlr5932 の大腸菌発現用プラスミド(pUC19acds) の構造を図９に示す。このアミノ酸コード領域の転写は lac プロモーターの制御下にあるように配置された。
Example 4: Construction and introduction of Agrobacterium expression vector Plasmid pUC19acds was extracted from E. coli BL21 (pUC19acds) carrying this plasmid according to a conventional method. FIG. 9 shows the structure of a plasmid for expression of Escherichia coli (pUC19acds) of mlr5932 derived from Mesorhizobium loti in which the amino acid coding region of mlr5932 is linked to plasmid vector pUC19. The transcription of this amino acid coding region was arranged to be under the control of the lac promoter.
M.loti ACCデアミナーゼ遺伝子（mlr5932）を制限酵素HindIIIとKpnIで切り出し、広宿主域ベクターpFAJ1709(Dombrecht et al., 2000)にサブクローニングした。M. loti 由来ACCデアミナーゼ遺伝子mlr5932 の広宿主域発現用プラスミド広宿主域プラスミドベクターpFAJ1709に、ACCデアミナーゼ遺伝子mlr5932のアミノ酸コード領域を結合させたプラスミド(pFAJacds)の構造を図１０に示す。このプラスミドにおいて、アミノ酸コード領域の転写はカナマイシン耐性遺伝子プロモーターの制御下にあるように配置された。図１０において、Ampはアンピシリン耐性遺伝子を、tetAとtetRはテトラサイクリン耐性遺伝子を、oriV:は複製開始点を、trfAは複製開始タンパク質を、それぞれ示す。 The M.loti ACC deaminase gene (mlr5932) was excised with restriction enzymes HindIII and KpnI and subcloned into a broad host range vector pFAJ1709 (Dombrecht et al., 2000). FIG. 10 shows the structure of a plasmid (pFAJacds) in which the amino acid coding region of the ACC deaminase gene mlr5932 is linked to the broad host range plasmid vector pFAJ1709 for expression of the M. loti-derived ACC deaminase gene mlr5932. In this plasmid, the transcription of the amino acid coding region was arranged to be under the control of the kanamycin resistance gene promoter. In FIG. 10, Amp represents an ampicillin resistance gene, tetA and tetR represent a tetracycline resistance gene, oriV: represents a replication origin, and trfA represents a replication initiation protein.
このプラスミドを保持する大腸菌BL21(pFAJacds)を、抗生物質アンピシリン100 mg/Lとテトラサイクリン1 mg/Lを含むLB培地で培養して維持した。ACCを添加した BL21(pFAJacds)株の粗酵素抽出液におけるα-ケト酪酸の蓄積速度（黒カラム）を実施例３の方法に従って測定したところ、対照としたBL21(pFAJ1709)株におけるα-ケト酪酸の蓄積速度（白カラム）に比べて大きくなり、このプラスミドが大腸菌BL21株にACCデアミナーゼ活性を付与出来ることが示された（図１１）。図１１においてカラムとエラーバーはそれぞれ、平均値と標準偏差を示している。
E. coli BL21 (pFAJacds) carrying this plasmid was cultured and maintained in LB medium containing antibiotics ampicillin 100 mg / L and
（実施例５）アグロバクテリウム菌へのACCデアミナーゼ遺伝子の導入と活性測定
続いて、このプラスミドpFAJ1709をA. tumefaciens C58C1RifR(pIG121-Hm)にエレクトロポレーション法により導入した。このプラスミドを保持する菌株C58C1RifR(pIG121-Hm, pFAJacds)は、抗生物質カナマイシン300 mg/L、アンピシリン300 mg/Lとテトラサイクリン50 mg/Lを含むLB培地で培養して維持した。対照とするプラスミドベクターpFAJ1709をもつ菌株C58C1RifR(pIG121-Hm, pFAJ1709)は、同様の方法により得た。２種類のプラスミドを同時に保持することの確認は、コロニーを鋳型とする直接 PCR法により確認したM. loti 由来ACCデアミナーゼ遺伝子mlr5932 の検出にはプライマーacds-Fとacds-R、uidAの検出にはプライマー35S-20F(5'-CGGGGGACTCTAGAGGATCC-3')とGUS+516R(5'-TGGTGTAGAGCATTACGCTG-3')をそれぞれ用いた。
(Example 5) Introduction of ACC deaminase gene into Agrobacterium and measurement of activity Subsequently, this plasmid pFAJ1709 was introduced into A. tumefaciens C58C1RifR (pIG121-Hm) by electroporation. The strain C58C1RifR (pIG121-Hm, pFAJacds) carrying this plasmid was maintained by culturing in an LB medium containing the antibiotics kanamycin 300 mg / L,
図１２にこの実験に使用したプラスミドの構造を示す。図１２の上段は対照とする遺伝子導入ヘルパープラスミド pIG121-Hm の構造を示す図である。P と T はそれぞれ、35S プロモーター、NOS ターミネーターを示している。また図１２の下段はmlr5932を有するプラスミド pFAJacds の構造を示す図であり、P と T はそれぞれ、カナマイシン耐性遺伝子 nptII のプロモーター、トリプトファン合成遺伝子 trpA のターミネーターを示している。更に選抜したアグロバクテリウム菌の細胞を鋳型としてPCR を行うことにより、２つのプラスミドの保持を確認した（図１３）。黒と白のアローはそれぞれ、構造遺伝子と PCR プライマー領域を示している。 FIG. 12 shows the structure of the plasmid used in this experiment. The upper part of FIG. 12 shows the structure of the gene introduction helper plasmid pIG121-Hm as a control. P and T indicate 35S promoter and NOS terminator, respectively. The lower part of FIG. 12 is a diagram showing the structure of plasmid pFAJacds having mlr5932, and P and T represent the kanamycin resistance gene nptII promoter and the tryptophan synthesis gene trpA terminator, respectively. Furthermore, by carrying out PCR using the selected Agrobacterium cells as a template, it was confirmed that the two plasmids were retained (FIG. 13). The black and white arrows indicate the structural gene and the PCR primer region, respectively.
更に獲得した菌株の粗酵素抽出液において、ACC 添加後のα-ケト酪酸の蓄積を観察した。プラスミド pFAJacds を保持するA. tumefaciens C58C1RifR(pIG121-Hm, pFAJacds)、および対照とした菌株 C58C1RifR(pIG121-Hm, pFAJ1709)の粗酵素抽出液において、ACC 添加後のα-ケト酪酸の蓄積速度を測定した（図１４）。カラムとエラーバーはそれぞれ、平均値と標準偏差を示している。 Furthermore, accumulation of α-ketobutyric acid after addition of ACC was observed in the obtained crude enzyme extract of the strain. Measure the accumulation rate of α-ketobutyric acid after addition of ACC in crude enzyme extracts of A. tumefaciens C58C1RifR (pIG121-Hm, pFAJacds) carrying plasmid pFAJacds and control strain C58C1RifR (pIG121-Hm, pFAJ1709) (FIG. 14). Columns and error bars show the mean and standard deviation, respectively.
菌株C58C1RifR(pIG121-Hm, pFAJacds)の粗酵素抽出液におけるα-ケト酪酸の蓄積速度（黒カラム）は、対照とした菌株C58C1RifR(pIG121-Hm, pFAJ1709)のα-ケト酪酸の蓄積速度（白カラム）に比べて大きくなった。このことは、本実験によりACC デアミナーゼ酵素活性をもつA. tumafaciensが作製できたことを示す。 The accumulation rate of α-ketobutyric acid in the crude enzyme extract of the strain C58C1RifR (pIG121-Hm, pFAJacds) (black column) is the accumulation rate of α-ketobutyric acid in the control strain C58C1RifR (pIG121-Hm, pFAJ1709) (white) Column). This indicates that A. tumafaciens having ACC deaminase enzyme activity could be produced by this experiment.
（実施例６）アグロバクテリウム菌へリゾビトキシン生産能力を付与する実験
ダイズ根粒菌Bradyrhizobium elkanii USDA94株のリゾビトキシンオペロンを含むコスミドpRTF1 (Yasuta et al. 2001)を制限酵素NheIにて切り出し、rtxAからORF4を含む約9 kbの断片を得た。この断片を広宿主域ベクターpBBR1MCS-2 (Kovach et al. 1995)のSpeIサイトへ、遺伝子がlacプロモーター転写制御下にあるように導入した。
(Example 6) Experiment for imparting lysovitoxin production ability to Agrobacterium cosmid pRTF1 (Yasuta et al. 2001) containing the lysovitoxin operon of the soybean rhizobia Bradyrhizobium elkanii USDA94 strain was cut out with restriction enzyme NheI and extracted from rtxA An approximately 9 kb fragment containing ORF4 was obtained. This fragment was introduced into the SpeI site of the broad host range vector pBBR1MCS-2 (Kovach et al. 1995) so that the gene was under the transcriptional control of the lac promoter.
図１５に、A. tumefaciens C58C1RifRへ導入したプラスミドの構造を示す。B. elkanii USDA94株のリゾビトキシンオペロンを含むコスミドpRTF1 (Yasuta et al. 2001)を制限酵素NheIにて切り出し、べクターpBBR1MCS-2に導入した。この導入した遺伝子の転写はlacプロモーターの制御下にあるように配置された。Kmrはカナマイシン耐性遺伝子を示している。そして、得られたプラスミドpBBR::PlacRTをA. tumefaciens C58C1RifR株へエレクトロポレーション法にて導入し、抗生物質カナマイシンによって導入された菌株を選択した。 FIG. 15 shows the structure of the plasmid introduced into A. tumefaciens C58C1RifR. Cosmid pRTF1 (Yasuta et al. 2001) containing the lysobitoxin operon of B. elkanii USDA94 strain was excised with restriction enzyme NheI and introduced into vector pBBR1MCS-2. The transcription of the introduced gene was arranged to be under the control of the lac promoter. Km r indicates a kanamycin resistance gene. Then, the obtained plasmid pBBR :: PlacRT was introduced into the A. tumefaciens C58C1RifR strain by electroporation, and a strain introduced by the antibiotic kanamycin was selected.
獲得した菌株 A. tumefaciens C58C1RifR (pBBR::PlacRT)を抗生物質カナマイシン 100 mg/Lを含むTris-YMRT液体培地 (Minamisawa et al. 1990) で2日間培養を行った。得られた菌体をリン酸カリウムバッファーに懸濁し、ただちに超音波破砕機にて菌体破砕を行ない、粗酵素抽出液を調製した。この粗酵素抽出液をサンプルに抗RtxA抗体を用いたウエスタンブロッティング解析を行い、プラスミドpBBR::PlacRTを保持するA. tumefaciens C58C1RifRにおけるrtxA遺伝子の発現を解析した。 The obtained strain A. tumefaciens C58C1RifR (pBBR :: PlacRT) was cultured in a Tris-YMRT liquid medium (Minamisawa et al. 1990) containing 100 mg / L of the antibiotic kanamycin for 2 days. The obtained microbial cells were suspended in a potassium phosphate buffer and immediately crushed by an ultrasonic crusher to prepare a crude enzyme extract. The crude enzyme extract was subjected to Western blotting analysis using an anti-RtxA antibody, and the expression of the rtxA gene in A. tumefaciens C58C1RifR carrying the plasmid pBBR :: PlacRT was analyzed.
図１６は、プラスミドpBBR::PlacRTおよびベクターとして使用したpBBR1MCS-2を保持するそれぞれのA. tumefaciens C58C1RifRの粗酵素抽出液をサンプルに、抗RtxA抗体を用いたウエスタンブロッティング解析の結果を示す。コントロールとしてB. elkanii USDA94 の野生株およびrtxAを含む広い遺伝子領域が欠失されている変異株 (Δrtx::Ω1)の粗酵素抽出液の結果を同時に示した。図１６より、A. tumefaciens C58C1RifR (pBBR::PlacRT)が有するrtxA遺伝子がIPTG添加の有無に関わらず大量に発現されることが明らかとなった（図１６：中央PlacRTのレーン）。 FIG. 16 shows the results of Western blotting analysis using an anti-RtxA antibody using a crude enzyme extract of each A. tumefaciens C58C1RifR carrying plasmid pBBR :: PlacRT and pBBR1MCS-2 used as a vector. As a control, the results of a crude enzyme extract of a wild strain of B. elkanii USDA94 and a mutant strain (Δrtx :: Ω1) from which a large gene region containing rtxA was deleted were shown simultaneously. FIG. 16 revealed that the rtxA gene possessed by A. tumefaciens C58C1RifR (pBBR :: PlacRT) was expressed in large quantities regardless of the presence or absence of IPTG addition (FIG. 16: lane of central PlacRT).
続いて、同様の菌株を抗生物質カナマイシン 100 mg/Lを含むTris-YMRT液体培地にて7日間培養を行い、その培養上清液のリゾビトキシンを含むアミノ画分をイオン交換樹脂にて精製した。次にPITC (フェニルイソチオシアネート)誘導体化を行いLC/MS解析分析を行った (Yasuta et al. 2001)。LC/MS解析によるA. tumefaciens C58C1RifR (pBBR::PlacRT)の培養上清に含まれるリゾビトキシンを検出した結果を図１７に示す。 Subsequently, the same strain was cultured in a Tris-YMRT liquid medium containing 100 mg / L of the antibiotic kanamycin for 7 days, and the amino fraction containing lysobitoxin in the culture supernatant was purified with an ion exchange resin. Next, PITC (phenylisothiocyanate) derivatization was performed and LC / MS analysis was performed (Yasuta et al. 2001). FIG. 17 shows the results of detecting lysobitoxin contained in the culture supernatant of A. tumefaciens C58C1RifR (pBBR :: PlacRT) by LC / MS analysis.
図１７のAは、PITCリゾビトキシン標準物質のスペクトルである。また図１７のBは、A. tumefaciens C58C1RifR (pBBR::PlacRT)の培養上清に対しPITC誘導体化を施した物質のスペクトルを示している。PITCリゾビトキシンの親イオン (M+1, m/z=461.3)およびフラグメントイオン (m/z=235.1)がA. tumefaciens C58C1RifR (pBBR::PlacRT)の培養上清にも検出された。即ち、PITCリゾビトキシン特有のスペクトルが検出された。この結果から、したがって、A. tumefaciens C58C1RifR (pBBR::PlacRT)はリゾビトキシンを生産し、アグロバクテリウム菌への生産能付与に成功したことが明らかとなった。 A of FIG. 17 is a spectrum of a PITC lysobitoxin standard substance. FIG. 17B shows the spectrum of a substance obtained by subjecting the culture supernatant of A. tumefaciens C58C1RifR (pBBR :: PlacRT) to PITC derivatization. PITC lysobitoxin parent ion (M + 1, m / z = 461.3) and fragment ion (m / z = 235.1) were also detected in the culture supernatant of A. tumefaciens C58C1RifR (pBBR :: PlacRT). That is, a spectrum peculiar to PITC lysobitoxin was detected. From this result, it was therefore clarified that A. tumefaciens C58C1RifR (pBBR :: PlacRT) produced lysobitoxin and succeeded in conferring production ability to Agrobacterium.
（実施例７）作出したアグロバクテリウム菌の遺伝子導入能力の評価
ACCデアミナーゼ活性を持ったA.tumefaciens C58C1RifR (pIG121-Hm, pFAJacds) の植物への遺伝子導入能力を評価した。外植片への遺伝子導入能力の評価は、実施例１に従い外植片の粗酵素抽出液のβ-グルクロニダーゼ酵素活性を利用した。
(Example 7) Evaluation of gene introduction ability of produced Agrobacterium
The ability of A. tumefaciens C58C1Rif R (pIG121-Hm, pFAJacds) having ACC deaminase activity to be introduced into plants was evaluated. Evaluation of gene introduction ability into explants was performed using β-glucuronidase enzyme activity of the crude enzyme extract of explants according to Example 1.
菌株C58C1RifR (pIG121-Hm, pFAJacds)とその対照区となる菌株C58C1RifR (pIG121-Hm, pFAJ1709)およびC58C1RifRは、28℃で液体LB培地を用いて、36時間震盪培養した。集められた菌体細胞は、MS液体培地で洗浄後、MS液体培地に懸濁した。この細胞懸濁液を107 cells/mLに調整して、メロンの子葉外植切片に接種した。メロンの外植切片は各処理区それぞれ15枚ずつ用意した。接種したメロン外植片は４日間共存培養した。共存培養4日目のβ-グルクロニダーゼ酵素の活性を測定した。 Strain C58C1Rif R (pIG121-Hm, pFAJacds) and its control strains C58C1Rif R (pIG121-Hm, pFAJ1709) and C58C1Rif R were subjected to shaking culture at 28 ° C. in a liquid LB medium for 36 hours. The collected bacterial cells were washed with MS liquid medium and then suspended in MS liquid medium. The cell suspension was adjusted to 10 7 cells / mL and inoculated into melon cotyledon explants. 15 explant sections of melon were prepared for each treatment section. The inoculated melon explants were co-cultured for 4 days. The activity of β-glucuronidase enzyme on the 4th day of co-culture was measured.
図１８は、A.tumefaciens C58C1RifR、C58C1RifR(pIG121-Hm, pFAJacds)、C58C1RifR(pIG121-Hm, pFAJ1709)を接種したメロン子葉外植片において、β-グルクロニダーゼの酵素活性を測定した結果を示すグラフである(接種後４日目)。１本のカラムは外植片１枚から抽出された本酵素の活性を示している。各処理区それぞれ15枚のメロンの子葉切片を供試した。 FIG. 18 shows the results of measuring the enzyme activity of β-glucuronidase in melon cotyledon explants inoculated with A. tumefaciens C58C1Rif R , C58C1Rif R (pIG121-Hm, pFAJacds) and C58C1Rif R (pIG121-Hm, pFAJ1709). It is a graph which shows (the 4th day after inoculation). One column shows the activity of the enzyme extracted from one explant. Fifteen melon cotyledon sections were used for each treatment group.
その結果、菌株C58C1RifR(pIG121-Hm, pFAJacds) 接種処理区（右カラム）では、C58C1RifRの接種処理区（左カラム）及びC58C1RifR(pIG121-Hm, pFAJ1709)接種処理区（中央カラム）に比べて、より高いβ-グルクロニダーゼ酵素活性が認められた。このことは、ACCデアミナーゼ合成能力を付与したアグロバクテリウム菌が同能力を有しないアグロバクテリウム菌に対して高い遺伝子導入能力を有することを示す。 As a result, in the C58C1Rif R (pIG121-Hm, pFAJacds) inoculation treatment (right column), the C58C1Rif R inoculation treatment (left column) and C58C1Rif R (pIG121-Hm, pFAJ1709) inoculation treatment (central column) In comparison, higher β-glucuronidase enzyme activity was observed. This indicates that Agrobacterium imparted with the ability to synthesize ACC deaminase has a higher gene transfer ability than Agrobacterium that does not have this ability.
本発明において提供されたアグロバクテリウム菌を使用して植物の形質転換を行うことにより、植物に遺伝子を効率的に導入することが可能である。例えば、従来から遺伝子導入が報告されている植物種においては、組換え植物の作出効率を向上させることができる。更には、本発明におけるアグロバクテリウム菌により、従来から遺伝子導入が困難とされていた植物種においても組換え植物の作出が可能になる。これにより、広い範囲の植物種で遺伝子組換えを利用した品種の改良が可能になる。 It is possible to efficiently introduce a gene into a plant by transforming the plant using the Agrobacterium provided in the present invention. For example, in plant species for which gene transfer has been reported, the production efficiency of recombinant plants can be improved. Furthermore, the Agrobacterium in the present invention makes it possible to produce recombinant plants even in plant species that have been conventionally difficult to introduce genes. This makes it possible to improve varieties using genetic recombination in a wide range of plant species.
（参考文献）
1. Yasuta, T., S. Okazaki, H. Mitsui, K. Yuhashi, H. Ezura and K. Minamisawa. 2001. DNA sequence and mutational analysis of rhizobitoxine biosynthesis gene in Bradyrhizobium elkanii. Appl. Environ. Microbiol. 67: 4999−5009.
2. Minamisawa, K., K. Fukai, and T. Asami. 1990. Rhizobitoxine inhibition of hydrogenase synthesis in free-living Bradyrhizobium japonicum. J. Bacteriol. 172: 4505-4509
3. Kovach, M. E., P. H.Elzer, D. S. Hill, G. T. Robertson, M. A. Farris, R. M. Roop II and K. M. Peterson. 1995. Four new derivatives of the broad-host-range cloning vector pBBR1MCS, carrying different antibiotic-resistance cassettes. 166: Gene 175-176.
4. Akama, K., Shiraishi, H., Ohta, S., Nakamura, K., Okada, K. and Shimura, Y. 1992. Efficient transformation of Arabidopsis thaliana : comparison of the efficiencies with various organs, plant ecotypes and Agorbacterium strains. Plant Cell Rep. 12 : 7-11.
5. Cardoza, V. and Stewart, C.N. 2003. Increased Agrobacterium-mediated transformation and rooting efficiencies in canola (Brassica napus L.) from hypocotyl segment explants. Plant Cell Rep. 21 : 599-604.
6. Cheng, M., Fry, J.E., Pang, S., Zhou, H., Hironaka, C.M., Duncan, D.R., Conner, T.W. and Wan, Y. 1997. Genetic transformation of wheat mediated by Agrobacterium tumefaciens. Plant Physiol. 115 : 971-980.
7. Cole M.A. and Elkan G.H. 1973. Transmissible resistance to penicillin G, neomycin, and chloramphenicol in Rhizobium japonicum. Antimicrob. Agents. Chemother. 4 : 248-253.
8. Dombrecht, B., Vanderleyden, J. and Michiels, J. 2001. Stable RK2-derived cloning vectors for the analysis of gene expression and gene function in gram negative bacteria. Mol. Plant-Microbe Interact. 14 : 426-430.
9. Ezura, H., Yuhashi, K., Yasuta, T. and Minamisawa, K. 2000. Effect of ethylene on Agrobacterium tumefaciens-mediated gene transfer to melon. Plant Breed. 119 : 75-79.
10. Fromm, M., Taylor, L.P. and Walbot, V. 1985. Expression of genes transferred into monocot and dicot plant cells by electroporation.Proc. Natl. Acad. Sci. USA. 82 : 5824-8.
11. Hanson, B., Engler, D., Moy, Y., Newman, B., Ralston, E. and Gutterson, N. 1999. A simple method to enrich an Agrobacterium-transformed population for plants containing only T-DNA sequences. Plant J. 19 : 727-734.
12. Hiei, Y., Ohta, S., Komori, T. and Kumashiro, T. 1994. Efficient transformation of rice (Oryza sativa L.) mediated by Agrobacterium and sequence analysis of the boundaries of the T-DNA. Plant J. 6 : 271-282.
13. Honma, M. and Shimomura, T. 1978. Metabolism of 1-amino-cyclopropane-1-carboxylic acid. Agri. Biol. Chem. 42 : 1825-1831.
14. Horlemann, C., Schwekendiek, A., Hohnle, M. and Weber, G. 2003. Regeneration and Agrobacterium-mediated transformation of hop (Humulus Iupulus L.) Plant Cell Rep. 22 : 210-217.
15. Murashige, T. and Skoog, F. 1962. A revised mediun for rapid growth and bioassay with tobacco tissue cultures. Physiol. Plant. 15 : 473-497.
16. Ohyama, A., Ito, H., Sato, T., Nishimura, S., Imai, T., Hirai, M. 1995. Supression of acid invertase activity by antisense RNA modifies the sugar composition of tomato fruit. Plant and Cell Physiology 36 (2): 369-376
17. Reitman, S. and Frankel, S. 1957. A colorimetric method for the determination of serum glutamic oxalacetic and glutamic pyruvic transaminases. Am. J. Clin. Pathol. 28 : 56-63.
18. Sambrook, J. and Russell, D.W. 2001. Molecular Cloning: a laboratory manual, 3rd edition. Cold spring harbor laboratory press, Cold spring harbor, N.Y.
19. Sheehy, R.E., Honma, M., Yamada, M., Sasaki, T., Martineau, B. and Hiatt, W.R. 1991. Isolation, sequence, and expression in Escherichia coli of the Pseudomonas sp. strain ACP gene encoding 1-aminocyclopropane-1-carboxylate deaminase. J. Bacteriol. 173 : 5260-5265.
20. Sunilkumar, G. and Rathore, K.S. 2001. Transgenic cotton: factors influencing Agrobacterium-mediated transformation and regeneration. Molecular Breeding 8 (1): 37-52
21. Van Larebeke N, Engler G, Holsters M, Van den Elsacker S, Zaenen I, Schilperoort RA, Schell J.Large plasmid in Agrobacterium tumefaciens essential for crown gall-inducing ability.Nature. 1974 Nov 8;252 (5479) :169-70.
22. Zupan, J.R. and Zambryski, P. 1995. Transfer of T-DNA from Agrobacterium to the plant cell. Plant Physiol. 107 : 1041-1047.
(References)
1. Yasuta, T., S. Okazaki, H. Mitsui, K. Yuhashi, H. Ezura and K. Minamisawa. 2001. DNA sequence and mutational analysis of rhizobitoxine biosynthesis gene in Bradyrhizobium elkanii. Appl. Environ. Microbiol. 67: 4999−5009.
2. Minamisawa, K., K. Fukai, and T. Asami. 1990. Rhizobitoxine inhibition of hydrogenase synthesis in free-living Bradyrhizobium japonicum. J. Bacteriol. 172: 4505-4509
3. Kovach, ME, PHElzer, DS Hill, GT Robertson, MA Farris, RM Roop II and KM Peterson. 1995. Four new derivatives of the broad-host-range cloning vector pBBR1MCS, carrying different antibiotic-resistance cassettes.166: Gene 175-176.
4. Akama, K., Shiraishi, H., Ohta, S., Nakamura, K., Okada, K. and Shimura, Y. 1992. Efficient transformation of Arabidopsis thaliana: comparison of the efficiencies with various organs, plant ecotypes and Agorbacterium strains. Plant Cell Rep. 12: 7-11.
5. Cardoza, V. and Stewart, CN 2003. Increased Agrobacterium-mediated transformation and rooting efficiencies in canola (Brassica napus L.) from hypocotyl segment explants.Plant Cell Rep. 21: 599-604.
6. Cheng, M., Fry, JE, Pang, S., Zhou, H., Hironaka, CM, Duncan, DR, Conner, TW and Wan, Y. 1997. Genetic transformation of wheat mediated by Agrobacterium tumefaciens. Plant Physiol 115: 971-980.
7. Cole MA and Elkan GH 1973. Transmissible resistance to penicillin G, neomycin, and chloramphenicol in Rhizobium japonicum. Antimicrob. Agents. Chemother. 4: 248-253.
8. Dombrecht, B., Vanderleyden, J. and Michiels, J. 2001. Stable RK2-derived cloning vectors for the analysis of gene expression and gene function in gram negative bacteria. Mol. Plant-Microbe Interact. 14: 426-430 .
9. Ezura, H., Yuhashi, K., Yasuta, T. and Minamisawa, K. 2000. Effect of ethylene on Agrobacterium tumefaciens-mediated gene transfer to melon. Plant Breed. 119: 75-79.
10. Fromm, M., Taylor, LP and Walbot, V. 1985. Expression of genes transferred into monocot and dicot plant cells by electroporation. Proc. Natl. Acad. Sci. USA. 82: 5824-8.
11. Hanson, B., Engler, D., Moy, Y., Newman, B., Ralston, E. and Gutterson, N. 1999. A simple method to enrich an Agrobacterium-transformed population for plants containing only T-DNA sequences. Plant J. 19: 727-734.
12. Hiei, Y., Ohta, S., Komori, T. and Kumashiro, T. 1994. Efficient transformation of rice (Oryza sativa L.) mediated by Agrobacterium and sequence analysis of the boundaries of the T-DNA. 6: 271-282.
13. Honma, M. and Shimomura, T. 1978. Metabolism of 1-amino-cyclopropane-1-carboxylic acid. Agri. Biol. Chem. 42: 1825-1831.
14. Horlemann, C., Schwekendiek, A., Hohnle, M. and Weber, G. 2003.Regeneration and Agrobacterium-mediated transformation of hop (Humulus Iupulus L.) Plant Cell Rep. 22: 210-217.
15. Murashige, T. and Skoog, F. 1962. A revised mediun for rapid growth and bioassay with tobacco tissue cultures. Physiol. Plant. 15: 473-497.
16. Ohyama, A., Ito, H., Sato, T., Nishimura, S., Imai, T., Hirai, M. 1995. Supression of acid invertase activity by antisense RNA modifies the sugar composition of tomato fruit. and Cell Physiology 36 (2): 369-376
17. Reitman, S. and Frankel, S. 1957. A colorimetric method for the determination of serum glutamic oxalacetic and glutamic pyruvic transaminases. Am. J. Clin. Pathol. 28: 56-63.
18. Sambrook, J. and Russell, DW 2001. Molecular Cloning: a laboratory manual, 3rd edition. Cold spring harbor laboratory press, Cold spring harbor, NY
19. Sheehy, RE, Honma, M., Yamada, M., Sasaki, T., Martineau, B. and Hiatt, WR 1991. Isolation, sequence, and expression in Escherichia coli of the Pseudomonas sp. Strain ACP gene encoding 1 -aminocyclopropane-1-carboxylate deaminase. J. Bacteriol. 173: 5260-5265.
20. Sunilkumar, G. and Rathore, KS 2001. Transgenic cotton: factors influencing Agrobacterium-mediated transformation and regeneration. Molecular Breeding 8 (1): 37-52
21. Van Larebeke N, Engler G, Holsters M, Van den Elsacker S, Zaenen I, Schilperoort RA, Schell J. Large plasmid in Agrobacterium tumefaciens essential for crown gall-inducing ability.Nature. 1974 Nov 8; 252 (5479): 169-70.
22. Zupan, JR and Zambryski, P. 1995. Transfer of T-DNA from Agrobacterium to the plant cell.Plant Physiol. 107: 1041-1047.
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