JP6826647B1 - DNA cassette constructs and methods for silencing phytoene synthase gene expression in specific plant tissues - Google Patents
DNA cassette constructs and methods for silencing phytoene synthase gene expression in specific plant tissues Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8216—Methods for controlling, regulating or enhancing expression of transgenes in plant cells
- C12N15/8218—Antisense, co-suppression, viral induced gene silencing [VIGS], post-transcriptional induced gene silencing [PTGS]
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- C—CHEMISTRY; METALLURGY
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- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
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- C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
- C12N15/8243—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine
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Abstract
【課題】特定の植物組織におけるフィトエンシンターゼ遺伝子発現をサイレンシングするためのDNA構築物およびその方法の提供。【解決手段】オンシジウムの花色を調節するDNAカセット構築体であって、幾つかのヌクレオチド配列から組み合わせられてなり、該DNAカセットは、植物の特定組織において、フィトエンシンターゼ遺伝子を干渉するmRNAを含み、花色調節の目的を達成し、該DNAカセットは、プロモーター及びヘアピン型干渉RNAを産生できるDNAヌクレオチド配列を含む、オンシジウムの花色を調節するDNAカセット。【選択図】図2PROBLEM TO BE SOLVED: To provide a DNA construct and a method thereof for silencing phytoene synthase gene expression in a specific plant tissue. A DNA cassette construct that regulates the flower color of oncidium, consisting of a combination of several nucleotide sequences, the DNA cassette containing mRNA that interferes with the phytoensynthase gene in a particular tissue of a plant. A DNA cassette that regulates the flower color of oncidium, which achieves the purpose of flower color regulation and contains a DNA nucleotide sequence capable of producing a promoter and hairpin-type interfering RNA. [Selection diagram] Fig. 2
Description
本発明は、フィトエンシンターゼ(phytoene synthase, PSY)遺伝子の発現をサイレンシング(silencing)し、オンシジウムの分野を制御してPSY遺伝子の発現を調整するヌクレオチド断片組成物及び方法に関し、特に、DNAカセット構築体に遺伝子移植することで特定の組織においてRNA干渉断片を生成し、従って、植物の特定の組織でのPSY遺伝子の発現はダウンレギュレート(Downregulate)することに関する。 The present invention relates to nucleotide fragment compositions and methods for silencing the expression of the phytoene synthase (PSY) gene and controlling the field of oncidium to regulate the expression of the PSY gene, in particular to construct a DNA cassette. Gene transplantation into the body produces RNA interfering fragments in specific tissues, thus relating to downregulate expression of the PSY gene in specific tissues of the plant.
オンシジウム(Oncidium)は、台湾の主要な輸出切り花の1つであり、台湾のオンシジウムの切り花の生産は、85%が他の国に輸出されており、日本は、台湾のオンシジウムの切り花の主要な輸出市場であり、台湾のオンシジウムは、日本のオンシジウムの総輸入量の第一位でもある。 Oncidium is one of Taiwan's major cut flowers for export, 85% of Taiwan's production of Oncidium cut flowers is exported to other countries, and Japan is the major cut flower for Taiwan's Oncidium. It is an export market, and Taiwan's Oncidium is also the number one in Japan's total imports of Oncidium.
しかしながら、品種が単一であることが、現在オンシジウム産業の深刻な問題の1つであるので、本発明のビジョンは、多様化され、差異を有する品種を創造すること、例えば、多様化された花の色を開発し、異なる輸出国及び輸出市場の要求を満たすようにし、新たな広い市場を開拓することである。台湾のオンシジウムの切り花は、黄色の花を主とし、現在の主流品種は、何れもオンシデサ ガワーラムジー(Oncidesa Gower Ramsey)から組織培養で変異を得られている。しかし、オンシデサガワーラムジーは、遠縁交配(distant hybridization)によるものであって、遠縁交配で得られた第1ハイブリッド(hybrid)のゲノムに大きな変化を発生し、相同染色体は、ペアリングプロセス(Pairing process)中に不均衡更には部分的欠落(chromosome deletion)を発生し、オンシデサ ガワーラムジーが有性生殖を行えなくなり、何れも組織培養の形で苗木生産を行う必要があり、従来のハイブリダイゼーション(Hybridization)では多様化した新しい品種を生成することは困難であり、この現象を突破するために、本発明は、RNA干渉技術を利用し、遺伝子移植の方法により、異なる花の色の品種のオンシジウムを開発している。 However, since the single variety is currently one of the serious problems of the onsidium industry, the vision of the present invention is to create varieties that are diversified and differ, eg, diversified. Develop flower colors to meet the demands of different exporting countries and export markets and open up new wide markets. The cut flowers of Oncidium in Taiwan are mainly yellow flowers, and all of the current mainstream varieties have been mutated by tissue culture from Oncidesa Gower Ramsey. However, Onsidesagawa ramsey is due to distant hybridization, which causes major changes in the genome of the first hybrid obtained by distant hybridization, and the homologous chromosomes undergo a pairing process ( Imbalance and even partial deletion (chromosome deletion) occur during the pairing process), which prevents onsidesagawaramuji from sexually reproducing, both of which require seedling production in the form of tissue culture, and conventional hybridization. It is difficult to generate diversified new varieties by (Hybridization), and in order to break through this phenomenon, the present invention utilizes RNA interference technology and uses gene transplantation methods to produce varieties of different flower colors. We are developing oncidium.
色は、植物商品の重要な特徴の1つであり、高等植物の花色は主にベタレイン(betalains)、フラボノイド(flavonoids)、カロテノイド(carotenoids)などの色素によって生成される。オンシデサガワーラムジーの黄色いリップ(lip)部位は、カロテノイドで構成されており、主成分は、ビオラキサンチン(violaxanthin)、9−シス−ビオラキサンチン(9-cis-violaxanthin)、ネオキサンチン(neoxanthin)及び少量のルテイン(lutein)であり、花上方の赤褐色の斑点は、アントシアニン(anthocyanins)からなり、主要成分は、シアニジン(cyanidin)、デルフィニジン(delphinidin)、及び少量のペオニジン(peonidin)である。そのうち、植物のカロテノイド生合成メカニズムの中で、フィトエンシンターゼ(phytoene synthase, PSY)は、2つのゲラニルゲラニル二リン酸(geranylgeranyl diphosphate)を1つのフィトエン(phytoene)(オクタヒドロリコピン)に合成できる重要な律速酵素であり、カロテノイド生合成経路の最も上流の化合物であり、後続でフィトエン(phytoene)は、フィトエンデサチュラーゼ(phytoene desaturase)とζ−カロテンデサチュラーゼ(ζ-carotene desaturase)を介して不飽和化され、リコピン(lycopene)を形成し、下流酵素の更なる触媒化を経て、各種異なる生成物を形成する。従って、フィトエンシンターゼは、カロテノイド生成過程の重要な酵素の1つであり、フィトエンシンターゼ遺伝子(OgPSY)の発現を抑制することにより、関連するカロテノイドの生成を抑制し、花の色を調節できる。 Color is one of the important characteristics of plant products, and the flower color of higher plants is mainly produced by pigments such as betalains, flavonoids and carotenoids. The yellow lip site of Onsidesagawa ramsey is composed of carotenoids, the main components of which are violaxanthin, 9-cis-violaxanthin, neoxanthin. And a small amount of lutein, the reddish-brown spots above the flowers consist of anthocyanins, and the main components are cyanidin, delphinidin, and a small amount of peonidin. Among them, in the carotenoid biosynthesis mechanism of plants, phytoene synthase (PSY) is an important rate-determining factor capable of synthesizing two geranylgeranyl diphosphate into one phytoene (octahydrolycopene). An enzyme, the most upstream compound of the carotenoid biosynthetic pathway, followed by phytoene, which is unsaturated via phytoene desaturase and ζ-carotene desaturase and lycopene. It forms (lycopene) and undergoes further catalysis of downstream enzymes to form a variety of different products. Therefore, phytoene synthase is one of the important enzymes in the carotenoid production process, and by suppressing the expression of the phytoene synthase gene (OgPSY), the production of related carotenoids can be suppressed and the color of flowers can be regulated.
RNA干渉原理またはRNAiは、Fireらによって最初に発見され、彼等は二本鎖RNA(dsRNA)によってワーム(Caenorhabidites elegan)体内に注入され、ワーム体内の一部の遺伝子がブロックされていることを発見した。植物の場合、アンチセンス鎖RNAを使用して花の色素の生成径路を変更し、異なる糸の花を創造する事例があり、例えば、アンチセンス鎖RNAを使用してペチュニア ハイブリッド(Petunia hybrid)中のカルコンシンターゼ(chalcone synthase)(PhCHS)の遺伝子発現を抑制して白色ペチュニアを生成する;アンチセンス鎖RNAを使用してガーベラハイブリダ(Gerbera hybrida)のカルコンシンターゼ(CHS)遺伝子の発現を抑制して橙色のガーベラを作り出す等である。これまでのところ、RNAi干渉技術を使用してカロテノイドを調整して花の色を変える事例は、遺伝子導入した菊花において、CCD4をノックダウン(knockdown)することで白い菊を鮮明な黄色の花色に転換した事例のみが出現している。さらに、カロテノイドは植物の成長の重要な要素であるため、重度に欠乏すると、光合作用などの多くの成長と発達の問題を引き起こすため、適切なプロモーターを選択してカロテノイド色素形成メカニズムの中の触媒化酵素の遺伝子を駆動調節し、例えば、フィトエンデサチュラーゼ(phytoene desaturase)、リコピンε−シクラーゼ(Lycopene ε-cyclase)、リコピンβ−シクラーゼ(Lycopene β-cyclase)などの酵素遺伝子により、特定の遺伝子を特定の組織で特異的に発現させることができ、花色を変える目的を達成するのみであるが、依然として植物の正常な生長の生理機能を維持し、これは技術の重要な鍵である。 The principle of RNA interference or RNAi was first discovered by Fire et al., That they were injected into the worm (Caenorhabidites elegan) by double-stranded RNA (dsRNA), blocking some genes in the worm. discovered. In the case of plants, there are cases where antisense strand RNA is used to alter the pigmentation pathway of flowers to create flowers of different threads, for example, in Petunia hybrid using antisense strand RNA. Suppresses the expression of the chalcone synthase (PhCHS) gene to produce white petunias; uses antisense strand RNA to suppress the expression of the Gerbera hybrida chalcone synthase (CHS) gene. To produce orange gerbera, etc. So far, an example of using RNAi interference technology to adjust carotenoids to change the color of a flower is a gene-introduced chrysanthemum flower that knocks down CCD4 to turn a white chrysanthemum into a bright yellow flower color. Only converted cases have appeared. In addition, because carotenoids are an important component of plant growth, severe deficiency causes many growth and developmental problems, such as photosynthesis, so choosing the right promoter to catalyze into the carotenoid pigmentation mechanism. Drives and regulates genes for chemases, and identifies specific genes by enzyme genes such as phytoene desaturase, lycopene ε-cyclase, and lycopene β-cyclase. Although it can be specifically expressed in the tissues of the plant and only achieves the purpose of changing flower color, it still maintains the physiological function of normal plant growth, which is an important key to the technology.
本発明の目的は、特定のプロモーターを使用して干渉RNAフラグメントを生成し、オンジシウムの花のフィトエンシンターゼ遺伝子の発現を減衰または抑制し、カロテノイドの形成を減らし、それによって花の色を変えるという目的を達成することであり、これらの観賞植物での干渉RNA技術の使用は、異なる遺伝子及び植物種を調節するために使用された前述の事例とはまったく異なる。 An object of the present invention is to generate interfering RNA fragments using specific promoters to attenuate or suppress the expression of the phytoene synthase gene in ondysium flowers, reduce the formation of carotenoids, and thereby change the color of the flowers. The use of interfering RNA techniques in these ornamental plants is quite different from the previous cases used to regulate different genes and plant species.
本発明の好適実施例の主な目的は、オンシジウムの標的遺伝子フィトエンシンターゼの発現をサイレンシング(silencing)又は低減する方法を提供することであり、それは、オンシジウムのフィトエンシンターゼ遺伝子をサイレンシングできるDNAカセット構築体し、このDNAカセット構築体ヌクレオチド配列であり、該ヌクレオチド配列の転写されたRNA断片は、オンシジウムフィトエンシンターゼ遺伝子のメッセンジャーRNA(messenger RNA, mRNA)の含有量を調節することに用いることができ、従って、花の色、匂い、またはカロテノイド生成物を必要とする他の植物生理学的メカニズムの調節を達成する。 A main object of the preferred embodiments of the present invention is to provide a method of silencing or reducing the expression of onsidium target gene phytoensynthase, which is a DNA cassette capable of silencing the onsidium phytoensynthase gene. constructs body is the DNA cassette construct nucleotide sequences, transcribed RNA fragment of said nucleotide sequence, can be used to adjust the content of the messenger RNA oncidium phytoene synthase gene (messenger RNA, mRNA) It can, therefore, achieves regulation of flower color, odor, or other phytophysiological mechanisms that require carotenoid products.
上記の目的を達成するために、本発明のヌクレオチド配列によって転写生成されるmRNAは、フィトエンシンターゼmRNAを相補結合のターゲットとし、フィトエンシンターゼと結合して二本鎖RNAとなった後、フィトエンシンターゼ遺伝子のmRNAを干渉し、酵素にさらに変換することはできず、カロテノイドの生合成反応が影響を受ける。該ヌクレオチド配列は、プロモーターと干渉RNA断片を転写できるDNAカセット構築体。該プロモーターは、配列番号1のヌクレオチド配列を含む。 In order to achieve the above object, the mRNA transcribed by the nucleotide sequence of the present invention targets the phytoene synthase mRNA as a complementary binding target, binds to phytoene synthase to become double-stranded RNA, and then the phytoene synthase gene. It interferes with the mRNA of and cannot be further converted to enzymes, affecting the biosynthetic reaction of carotenoids. The nucleotide sequence is a DNA cassette construct capable of transcribing a promoter and an interfering RNA fragment. The promoter comprises the nucleotide sequence of SEQ ID NO: 1.
本発明の好適実施例は、DNAカセット構築体を提示し、このカセットは、プロモーター(P)を含み、その後に3’末端に配列番号3のDNA断片を接続し、更に、GUS遺伝子を含むイントロン断片(intronGUSヌクレオチド配列)、及び一方向に反転した配列番号3のDNA断片を接続し、最後に、NOS遺伝子のターミネーター断片(T−NOS ヌクレオチド配列)を接続する。上記の組換え色素体またはベクターには、特定のプロモーター、イントロン、またはターミネーター断片、または本発明での使用に適した他の組換え色素体またはベクターが含まれるが、これらに限定されない。ここで、ヌクレオチド配列は、プロモーターPchrcの後に配置され、ヌクレオチド配列は、それぞれ約150個のヌクレオチドの配列番号3のヌクレオチド断片の2つの小さな片を含み、両者は、相反方向の配列を呈し、中間にGUS−イントロンヌクレオチド断片を挟み、このヌクレオチド配列断片は、プロモーターPchrcの駆動の下でヘアピン構造のRNA分子を転写生成できる。このRNA断片は、標的mRNA、例えばフィトエンシンターゼ遺伝子などのmRNAを有し、これはヌクレオチド配列に相補的であり、二本鎖RNA構造を形成する。 A preferred embodiment of the present invention presents a DNA cassette construct, which contains a promoter (P), followed by a DNA fragment of SEQ ID NO: 3 at the 3'end, and an intron containing the GUS gene. The fragment (intronGUS nucleotide sequence) and the unidirectionally inverted DNA fragment of SEQ ID NO: 3 are connected, and finally, the terminator fragment of the NOS gene (T-NOS nucleotide sequence) is connected. The recombinant plastids or vectors described above include, but are not limited to, specific promoters, introns, or terminator fragments, or other recombinant plastids or vectors suitable for use in the present invention. Here, the nucleotide sequence is placed after the promoter Pchrc, and the nucleotide sequence contains two small pieces of the nucleotide fragment of SEQ ID NO: 3 of about 150 nucleotides each, both exhibiting sequences in opposite directions and intermediate. A GUS-intron nucleotide fragment is sandwiched between the two, and this nucleotide sequence fragment can transcribe an RNA molecule having a hairpin structure under the drive of the promoter Pchrc. This RNA fragment carries a target mRNA, such as an mRNA such as the phytoene synthase gene, which is complementary to the nucleotide sequence and forms a double-stranded RNA structure.
配列番号3は、配列番号2の51番目のヌクレオチドから150個のヌクレオチドの長さのヌクレオチド配列である。 SEQ ID NO: 3 is a nucleotide sequence having a length of 150 nucleotides from the 51st nucleotide of SEQ ID NO: 2.
本発明の好適実施例は、配列番号1、配列番号2、配列番号3を含むヌクレオチド配列、及び配列番号1、配列番号2、配列番号3の配列に相補的なヌクレオチド配列を提供する。 A preferred embodiment of the present invention provides a nucleotide sequence comprising SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 and a nucleotide sequence complementary to the sequences of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3.
本発明の好適実施例のヌクレオチド配列は、ベクターまたはシャトルベクター(shuttle vector)で提供される。 Nucleotide sequences of preferred examples of the invention are provided as vectors or shuttle vectors.
本発明の好適実施例のヌクレオチド配列は、プロトコーム様体(Protocorm-like body, PLB)で提供される。 The nucleotide sequences of preferred examples of the present invention are provided in a Protocorm-like body (PLB).
本発明の好適実施例のベクターは、組換えにより植物細胞またはプロトコーム様体(Protocorm-like body,PLB)に導入することができる。 The vector of the preferred embodiment of the present invention can be introduced into a plant cell or a Protocorm-like body (PLB) by recombination.
本発明の好適実施例のヌクレオチド配列は、植物組織で提供される。 The nucleotide sequences of preferred examples of the present invention are provided in plant tissue.
上記の目的を達成するために、本発明の好適実施例のオンシジウムのフィトエンシンターゼ遺伝子のダウンレギュレーション(down regulation)及びサイレンシング方法は、少なくとも1つのDNAカセット構築体を提供し、このDNAカセット構築体のヌクレオチド配列は、特定のプロモーター及びヘアピン型干渉RNA断片に転写可能な特定のヌクレオチド断片を含み、ヌクレオチド配列を植物細胞(またはPLB)に導入して、組換えた植物細胞(またはPLB)を取得し、組換えた植物細胞を使用して植物に再生し、植物の花色を変えるという目的を達成する。 In order to achieve the above object, the down regulation and silencing method of the oncidium phytoensynthase gene of the preferred embodiment of the present invention provides at least one DNA cassette construct, which is the DNA cassette construct. Nucleotide sequence contains a specific promoter and a specific nucleotide fragment that can be transcribed into a hairpin-type interfering RNA fragment, and the nucleotide sequence is introduced into a plant cell (or PLB) to obtain a recombinant plant cell (or PLB). Then, the recombinant plant cells are used to regenerate the plant to achieve the purpose of changing the flower color of the plant.
本発明の好適実施例のヌクレオチド配列は、遺伝子クローニング技術(gene cloning)により得られる。 The nucleotide sequence of a preferred example of the present invention is obtained by gene cloning technology.
本発明の好適実施例の組換えた植物細胞は、植物体内のフィトエンシンターゼの表現量を減衰するために使用される。 The recombinant plant cells of the preferred examples of the present invention are used to attenuate the expression of phytoene synthase in plants.
本発明の好適実施例は、組換えた植物細胞の再生を利用して組換えた植物細胞を製造する。 A preferred embodiment of the present invention utilizes the regeneration of recombinant plant cells to produce recombinant plant cells.
本発明の好適実施例の植物細胞は、ラン科植物細胞から選択される。 The plant cells of the preferred examples of the present invention are selected from Orchidaceae plant cells.
本発明の特定の植物組織におけるフィトエンシンターゼ遺伝子発現をサイレンシングするためのDNA構築物およびその方法は、特定のプロモーターを使用して干渉RNAフラグメント(fragment)を生成し、オンジシウムの花のフィトエンシンターゼ遺伝子の発現を減衰または抑制し、カロテノイドの形成を減らし、それによって花の色を変えることができる。 DNA constructs and methods for silencing phytoene synthase gene expression in specific plant tissues of the present invention use specific promoters to generate interfering RNA fragments of ondysium flower phytoene synthase genes. Expression can be attenuated or suppressed, reducing carotenoid formation, thereby altering flower color.
本発明を十分に理解するために、好適実施例を図面と併せて以下に例示し、且つそれは本発明を限定することを意図するものではない。 In order to fully understand the present invention, preferred examples are exemplified below together with the drawings, and it is not intended to limit the present invention.
本発明の好適実施例のオンシジウムのフィトエンシンターゼ遺伝子は、その遺伝子生成物(gene product)またはその番号のタンパク質を提供するのに適しているが、本発明の範囲を限定するものではない。さらに、本発明の好適実施例のオンシジウムのフィトエンシンターゼ遺伝子及びその調節方法は、各種遺伝子工程による各種植物細胞またはプロトコーム様体への遺伝子の導入に適しているが、本発明の範囲を限定するものではない。 The oncidium phytoene synthase gene of a preferred embodiment of the invention is suitable for providing the gene product or the protein of that number, but does not limit the scope of the invention. Further, the Oncidium phytoene synthase gene and the method for regulating the oncidium phytoene synthase gene of the preferred embodiment of the present invention are suitable for introducing the gene into various plant cells or protocomb-like bodies by various gene steps, but limit the scope of the present invention. is not it.
例えば、本発明の好適実施例で使用されるオンシジウムは、黄色の花の品種(たとえば、オンシデサハニーエンジェル)であるが、本発明は、その他の各種オンシジウムの品種に適用され、本発明の範囲を限定するものではない。 For example, the Oncidium used in the preferred embodiments of the present invention is a yellow flower variety (eg, Onsidesa Honey Angel), but the present invention applies to various other Oncidium varieties and is the scope of the present invention. Is not limited to.
本発明の好適実施例のオンシジウムのフィトエンシンターゼ遺伝子をサイレンシングする技術は、干渉RNAを産生するDNAカセット構築体であり、その構造説明図は、図2に示すとおりであり、ヘアピンRNAを嵌合する転移DNA(T−DNA)カセットを転写可能に構築され、ベクター中に組み合わせられ、このDNAカセット構築体は、1つのプロモーターを含み、その3’末端に配列番号3の正方向に安置されたヌクレオチド配列、及びイントロンGUSを含むイントロン断片(intronGUSヌクレオチド配列)を接続し、更に配列番号3の逆ヌクレオチド配列を接続し、更に、NOS遺伝子のターミネーター断片のT−NOSヌクレオチド配列を接続する。上記の組換え色素体またはベクターには、特定のプロモーターまたはイントロン(intron)断片、または本発明に適した他の組換え色素体またはベクターを含むが、これらに限定するものではない。 The technique for silencing the oncidium phytoensynthase gene of a preferred embodiment of the present invention is a DNA cassette construct that produces interfering RNA, the structural explanatory diagram of which is as shown in FIG. 2, and the hairpin RNA is fitted. The translocated DNA (T-DNA) cassette to be transcribed was constructed transcribably and combined into a vector, the DNA cassette construct containing one promoter, which was placed at its 3'end in the positive direction of SEQ ID NO: 3. The nucleotide sequence and the intron fragment containing intron GUS (intron GUS nucleotide sequence) are connected, the reverse nucleotide sequence of SEQ ID NO: 3 is further connected, and the T-NOS nucleotide sequence of the terminator fragment of the NOS gene is further connected. The recombinant plastids or vectors described above include, but are not limited to, specific promoters or intron fragments, or other recombinant plastids or vectors suitable for the present invention.
1.オンシデサハニーエンジェルからプロモーターを取得する。
この方法は、オンシデサハニーエンジェルの全RNAを、Oligotex mRNA Kit (Qiagen)を使用して、Poly(A)+RNAに精製する。Clontech製造業者が提供する操作方法に従って、高速増幅技術(Rapid Amplification of cDNA Ends, RACE)を利用し、cDNAの5’末端および3’末端から全長DNAを増幅し、オンシデサハニーエンジェルの色素体特異的カロテノイド結合タンパク質(chromoplast specific carotenoid associated protein, CHRC )遺伝子の完全長cDNAヌクレオチド配列を取得する。
本発明で使用されるプロモーター選択方法は、分子生物学的ゲノムウォーカー技術(GenomeWalker Universal Kit, Clontech)を使用して、CHRCcDNAからこの遺伝子プロモーター断片Pchrc、合計1704bpに増幅した(図1)。
1. 1. Obtain a promoter from the Onsidesa Honey Angel.
This method purifies the total RNA of Onsidesa Honey Angel into Poly (A) + RNA using the Oligotex mRNA Kit (Qiagen). According to the operating method provided by the Clontech manufacturer, full-length DNA is amplified from the 5'and 3'ends of cDNA using Rapid Amplification of cDNA Ends (RACE) and plastid-specific for Onsidesa Honey Angel. Obtain the full-length cDNA nucleotide sequence of the chromaplast specific carotenoid associated protein (CHRC) gene.
The promoter selection method used in the present invention was amplified from CHRC cDNA to a total of 1704 bp of this gene promoter fragment Pchrc using molecular biological genome walker technology (GenomeWalker Universal Kit, Clontech) (Fig. 1).
2.RNA干渉の標的遺伝子の選択。
NCBIデータベース内の4つの単子葉植物種(イネ、小麦、トウモロコシ、胡蝶蘭)のフィトエンシンターゼヌクレオチド配列を対照し、相同性分析を行い、150bpのフィトエンシンターゼ調節遺伝子のcDNA領域(オープンリーディングフレーム(open reading frame)で51bp〜+200bp)が、22−bpの100%相同性の保守配列が対応した相補的DNA(cDNA)配列に含み、RNA干渉のターゲットヌクレオシド配列として選択される。
2. 2. Selection of target genes for RNA interference.
Phytoene synthase nucleotide sequences of four monocotyledonous plant species (rice, wheat, corn, and butterfly orchid) in the NCBI database were controlled, homology analysis was performed, and the cDNA region of the 150 bp phytoene synthase regulatory gene (open reading frame (open) In the reading frame), 51 bp to +200 bp) is included in the corresponding complementary DNA (cDNA) sequence with a 100% homologous conservative sequence of 22-bp and is selected as the target nucleoside sequence for RNA interference.
3.遺伝子移植および培養方法。
本発明のDNAカセット構築体を含めてベクターに構築し、アグロバクテリウム法を利用してこのベクターを携帯してオンシデサハニーエンジェル(Onc. Honey Angel)のプロトコーム様体(Protocorm-like body, PLB)を感染させ、適切な濃度の抗生物質を含む培地を利用して移植後のプロトコーム様体をスクリーニングし、それから、シューティング(shooting)及びルーティング(rooting)の培地によって植物全体に誘導し、植物を鉢植えで2年、その花の色を観察した。上記の組換え法には、アグロバクテリウム媒体法、遺伝子組換えウイルス感染法、ジャンパーベクター導入法、遺伝子銃導入法、エレクトロポレーション、マイクロインジェクション法、花粉管法、脂質体媒体導入法、超音波媒体導入法、炭化ケイ素繊維媒体導入法等の当業者によく知られたものを含むが、これらに限定するものではない。蘭の花の組織培養の方法も本発明の当業者によく知られているため、再度説明しない。
3. 3. Gene transplantation and culture method.
The DNA cassette construct of the present invention was constructed into a vector, and the vector was carried by the Agrobacterium method to carry the Onc. Honey Angel Protocorm-like body (PLB). ) Is infected, the protocomb-like body after transplantation is screened using a medium containing an appropriate concentration of antibiotics, and then the whole plant is guided by a shooting (shooting) and rooting (rooting) medium to induce the plant. The color of the flower was observed for 2 years in a potted plant. The above recombinant methods include Agrobacterium medium method, recombinant virus infection method, jumper vector introduction method, gene gun introduction method, electroporation, microinjection method, pollen tube method, lipid body medium introduction method, and ultrasonic method. It includes, but is not limited to, those well known to those skilled in the art such as the ultrasonic medium introduction method and the silicon carbide fiber medium introduction method. The method of tissue culture of orchids is also well known to those skilled in the art of the present invention and will not be described again.
≪実施例1:オンシジウムの花色の調節≫
図3に示すように、左から右に、それぞれ野生型(WF)オンシデサハニーエンジェルの花、本発明のDNAカセット構築体ヌクレオチド配列が遺伝子組換え植物オンシデサMF−1、および本発明のDNAカセット構築体ヌクレオチド配列が遺伝子組換え植物オンシデサMF−5を示し、MF−1が淡白色の花色であり、MF−5が雪白色の花色であることが分かる。
<< Example 1: Adjustment of Oncidium flower color >>
As shown in FIG. 3, from left to right, wild-type (WF) onsidesa honey angel flowers, the DNA cassette construct nucleotide sequence of the present invention is a genetically modified plant onsidesa MF-1, and the DNA cassette of the present invention. It can be seen that the construct nucleotide sequence shows the transgenic plant Onsidesa MF-5, MF-1 has a pale white flower color, and MF-5 has a snow-white flower color.
製品の表現型の安定性を維持するために、本実施例では30ppmのハイグロマイシン(hygromycin)の半分の濃度(1/2)のムラシゲ&スクーグ(Murashige & Skoog (MS))を含む培地を使用して遺伝子組換え植物オンシデサMF−1及びオンシデサMF−5のP0段階の微小な分裂組織の芽を培養する。プロトコーム様体の分裂及び植物の分岐生長作用を経て、2つの系統(line)で2年間の栽培後にそれぞれP1苗を2000株生産して温室に植えた。苗の生長温度は30°C/25°C(昼/夜)であり、光合成光子束密度(PPFD)が約300μmol−2s−1の環境で光強度が制御される。 To maintain product phenotypic stability, this example uses medium containing half the concentration (1/2) of 30 ppm hygromycin (Murashige & Skoog (MS)). to culturing the buds P 0 stage of fine meristems of transgenic plants Onshidesa MF-1 and Onshidesa MF-5. Through the division and branch growth action of the plant of Protocorm-like bodies, each after two years of cultivation were planted in the greenhouse to produce 2000 shares of P 1 seedlings in two lines (line). The growth temperature of seedlings is 30 ° C / 25 ° C (day / night), and the light intensity is controlled in an environment where the photosynthetic photon bundle density (PPFD) is about 300 μmol -2 s- 1 .
実施例2:オンシジウムの花のカロテノイド含有量
図4に示すように、遺伝子組換え植物オンシデサMF−1及びオンシデサMF−5のカロテノイドは大幅に減少し、前述の両者の9−cis−ビオラキサンチン含有量は、いずれも野生型の40%であったが、オンシデサMF−1のネオキサンチン(neoxanthin)、ビオラキサンチン(violaxanthin)及びβ−カロチン(β-carotene)は、いずれも野生型の30%であった。純白色の花色を呈するオンシデサMF−5では、これら3つの色素の含有量は、何れも野生型の1〜3%のみであり、この結果は、組換え本発明のDNAカセットのヌクレオチド配列がカロテノイドの生成を有効に制御でき、花色を制御する効果を達成できることを示している。
Example 2: Carotenoid content of oncidium flowers As shown in FIG. 4, the carotenoids of the recombinant plants Onsidesa MF-1 and Onsidesa MF-5 were significantly reduced, and both of them contained 9-cis-violaxanthin as described above. The amount was 40% of the wild type, but neoxanthin, violaxanthin and β-carotene of Onsidesa MF-1 were all 30% of the wild type. there were. In Onsidesa MF-5, which has a pure white flower color, the content of all three pigments is only 1 to 3% of the wild type, and the result is that the nucleotide sequence of the recombinant DNA cassette of the present invention is a carotenoid. It is shown that the production of the flower can be effectively controlled and the effect of controlling the flower color can be achieved.
実施例3:オンシジウム組織中の遺伝子発現量
図5は、フィトエンシンターゼ遺伝子(OgPSY)の発現量を示しており、野生型のオンシジウムのOgPSY発現量が高く、遺伝子組換え植物オンシデサMF−1及びオンシデサMF−5は、バンドが見られず、その発現量は明らかに低減し、野生型は、依然としてOgPSYを発現していることが分かる。また、図6から、遺伝子組換え植物オンシデサMF−1およびオンシデサMF−5のsiRNA(小さな分子の干渉RNA)が強く発現しており、遺伝子発現から結果を判断できることを示している。これは、本発明がOgPSYの発現の干渉に成功し、色素と花色を制御する目的を達成することを示している。
Example 3: Gene expression level in oncidium tissue FIG. 5 shows the expression level of the phytoensynthase gene (OgPSY), the OgPSY expression level of wild-type oncidium is high, and the recombinant plants Onsidesa MF-1 and Onsidesa It can be seen that MF-5 does not show a band and its expression level is clearly reduced, and the wild type still expresses OgPSY. In addition, FIG. 6 shows that siRNA (interfering RNA of a small molecule) of the transgenic plants Onsidesa MF-1 and Onsidesa MF-5 is strongly expressed, and the result can be judged from the gene expression. This indicates that the present invention succeeds in interfering with the expression of OgPSY and achieves the purpose of controlling pigment and flower color.
Claims (7)
前記プロモーターは、ヌクレオチド配列番号1のヌクレオチド配列を含み、且つ該プロモーターは、特定遺伝子を特定の組織で特異的に発現させ、花色を変える目的のみを達成するが、依然として植物の正常な生長の生理学的機能を維持し、
一部のヘアピン型干渉RNAヌクレオチド配列を生成できるDNAヌクレオチド配列は、前記プロモーターの駆動の下、特定組織においてヘアピン型干渉RNA配列を転写して生成し、フィトエンシンターゼ遺伝子のmRNAを干渉し、酵素にさらに変換することはできず、正方向PSYヌクレオチド配列、イントロンGUSヌクレオチド配列、逆方向PSYヌクレオチド配列、およびNOS遺伝子のターミネーター断片(T−NOSヌクレオチド配列)で組成され、ここで、該PSYヌクレオチド配列は、配列番号3のヌクレオチド配列を含む、DNAカセット構築体。 A DNA cassette construct that regulates the flower color of oncidium, consisting of a combination of several nucleotide sequences, the DNA cassette construct contains an mRNA that interferes with the phytoensynthase gene in a particular tissue of a plant and regulates the flower color. The DNA cassette construct regulates the flower color of oncidium, which contains a promoter and a DNA nucleotide sequence capable of producing hairpin-type interfering RNA .
The promoter comprises the nucleotide sequence of nucleotide SEQ ID NO: 1, and the promoter specifically expresses a particular gene in a particular tissue to achieve only the purpose of altering flower color, but still the physiology of normal plant growth. Maintain the function
A DNA nucleotide sequence capable of generating some hairpin-type interfering RNA nucleotide sequences is produced by transcribing the hairpin-type interfering RNA sequence in a specific tissue under the drive of the promoter, interfering with the mRNA of the phytoensynthase gene, and becoming an enzyme. It cannot be further converted and is composed of a forward PSY nucleotide sequence, an intron GUS nucleotide sequence, a reverse PSY nucleotide sequence, and a terminator fragment of the NOS gene (T-NOS nucleotide sequence), wherein the PSY nucleotide sequence is , A DNA cassette construct comprising the nucleotide sequence of SEQ ID NO: 3 .
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