JP2004535810A - Plant ion channels and methods - Google Patents

Abstract

Provided are recombinant plant receptor proteins and nucleotide sequences encoding these proteins. The present invention also provides a recombinant vector comprising a nucleotide sequence encoding a protein. Transgenic plants and methods using the nucleotide and amino acid sequences described herein, including plant host cells containing recombinant vectors, methods of treating plants, expressing proteins described herein Methods, methods for altering plant receptor activity and methods for regulating plant metabolism are provided. It is believed that the plant receptor protein of the present invention functions as an effective modulator of the action of GABA.

Description

【００６９】
表１のデータは内因性ＧＡＢＡレベルのような、低レベルのＧＡＢＡが植物中に存在する場合、作用を確認するためには高濃度のＰＫ１１１９５が必要であることを示唆する。
図５に示すように、上記のように生長したウキクサをジアゼパムで独立して処理した場合、培地中のＧＡＢＡの存在下で生長促進作用が見られた。ウキクサをＰＫ１１１９５で独立して処理した場合、培地中のＧＡＢＡの存在下で生長阻害作用が見られた。
【００７０】
初期の研究は、ウキクサ生長に対する動物ＧＡＢＡ受容体アンタゴニストの作用を報告した；しかし、以下の表２で報告されたＧＡＢＡ生理活性の阻害剤は、初期の研究で使用されたＧＡＢＡ受容体アンタゴニストより最高で１０００倍まで活性が高かった。このことは、異なるクラスのＧＡＢＡ受容体に作用していること、および動物と同様に、植物も多様なＧＡＢＡ受容体を有することを示唆する。
【００７１】
【表２】

【００７２】
動物のベンゾジアゼピン受容体の活性を促進または阻害する薬剤を用いた実験が、植物でも類似した結果であったことから、上記の結果と合わせて、植物におけるベンゾジアゼピン、またはベンゾジアゼピン様受容体の証拠を提供する。
【実施例２】
【００７３】
全長ｃＤＮＡおよびゲノムＤＮＡの単離
プロトコール
シロイヌナズナＡｒａｂｉｄｏｐｓｉｓ ｔｈａｌｉａｎａ（Ｌ．）Ｈｅｙｎｈ．エコタイプＣｏｌｕｍｂｉａ（Ｃｏｌ−０）の種子はＡｒａｂｉｄｏｐｓｉｓ Ｂｉｏｌｏｇｉｃａｌ Ｒｅｓｏｕｒｃｅ Ｃｅｎｔｅｒ（Ｏｈｉｏ Ｓｔａｔｅ Ｕｎｉｖｅｒｓｉｔｙ，Ｃｏｌｕｍｂｕｓ，ＯＨ）から得ることができる。シロイヌナズナの苗は回転振とう機（１５０ｒｐｍ）上のＭＳ培地［Ｍｕｒａｓｈｉｇｅ ａｎｄ Ｓｋｏｏｋ，Ｐｈｙｓｉｏｌ．Ｐｌａｎｔ １５：４８５（１９６２）］を含むフラスコ中で無菌的に育てる。２日目の苗を総ＲＮＡ単離のために回収する。総ＲＮＡはＴｕｒａｎｏ，Ｆ．Ｊ．ｅｔ ａｌ．（１９９２）Ｐｌａｎｔ Ｐｈｙｓｉｏｌ．１００：３７４に記載のように単離する。合成されたプライマー
【００７４】
【化１】

【００７５】
および
【００７６】
【化２】

【００７７】
（それぞれＳＥＱ ＩＤ ＮＯ：３および４）（ＧｅｎＢａｎｋ，未知蛋白質、遺伝子＃Ａｔ２ｇ４７７７０，蛋白質 ｉｄ＝ＡＡＣ６３６３２．１，ｄｂ ｘｒｅｆ＝ＧＩ：３７３８２９０）は市販（Ｂｉｏｓｙｎｔｈｅｓｉｓ，Ｉｎｃ．Ｌｅｗｉｓｖｉｌｌｅ，ＴＸ）され、ＲＴ‐ＰＣＲ反応に使用される。ＲＴ‐ＰＣＲでは、５′ＲＡＣＥ系（Ｌｉｆｅ Ｔｅｃｈｎｏｌｏｇｉｅｓ，Ｒｏｃｋｖｉｌｌｅ，ＭＤ）を使用して、全長ｃＤＮＡクローンを同定する。プライマー３′ＥｃｏＰＢＲを使用して、取り扱い説明書に従い、２日目の植物から単離したポリ（Ａ⁺）ＲＮＡ１μｇ由来の第１鎖ｃＤＮＡを合成する。第１鎖ｃＤＮＡ合成物の１／５を、両プライマー、５′ＥｃｏＰＢＲおよび３′ＥｃｏＰＢＲによる遺伝子増幅反応における鋳型として使用する。増幅の前に、成分を９５℃で４分間インキュベートする。遺伝子増幅反応は、９４℃で１分間、６８℃で１分間および７２℃で２分間を３０サイクル行い、その後７２℃で５分間延長して行う。
【００７８】
ゲノムＤＮＡはＴｕｒａｎｏ，Ｆ．Ｊ．ｅｔ ａｌ．（１９９２）Ｐｌａｎｔ Ｐｈｙｓｉｌｏ．１００：３７４に記載のように、２４日目のシロイヌナズナの葉から単離する。ＰＣＲ反応では、それぞれのプライマー（５′ＥｃｏＰＢＲおよび３′ＥｃｏＰＢＲ） ２５０ｎｇとゲノムＤＮＡ ５００ｎｇを使用する。増幅反応の前に、成分を９５℃で１０分間インキュベートする。遺伝子増幅反応は、９４℃で１分間、７０℃で１分間および７２℃で３分間を３０サイクル行い、その後７２℃で５分間延長して行う。
【００７９】
ゲノムＤＮＡおよびｃＤＮＡフラグメントは別々にＰＣＲ２．１（Ｉｎｖｉｔｒｏｇｅｎ Ｃｏｒｐ．Ｃａｒｌｓｂａｄ，ＣＡ，ＵＳＡ）にクローニングし、Ｔａｑ Ｄｉｄｅｏｘｙ ｔｅｒｍｉｎａｔｏｒ ｃｙｃｌｅ ｓｅｑｕｅｎｃｅ（Ａｐｐｌｉｅｄ Ｂｉｏｓｙｓｔｅｍｓ）法により配列決定する。データはＰｏｗｅｒ Ｍａｃｉｎｔｏｓｈ ６５００／２５０上でＭａｃＶｅｃｔｏｒソフトウェアにより解析する。
【実施例３】
【００８０】
トランスジェニック植物の構築
植物受容体蛋白質を過剰発現するトランスジェニック植物、またはアンチセンス受容体蛋白質を過剰発現するものは以下のように作製する。ＰＣＲ、ＲＴ‐ＰＣＲまたはアンチセンス構築物を作製するための慣用のクローニング法を使用して、受容体蛋白質のセンス（過剰発現）またはアンチセンス（不十分な発現）の全（５９１塩基対）オープンリーディングフレーム、または約２５塩基対の大きさのその一部（アンチセンスまたはＲＮＡｉだけのための）をｐＢＩ１２１（Ｃｌｏｎｅｔｅｃｈ，Ｐａｌｏ Ａｌｔｏ，ＣＡ）のようなベクターにクローニングする。合成された遺伝子特異的プライマー、
【００８１】
【化３】

【００８２】
および
【００８３】
【化４】

【００８４】
（ＧｅｎＢａｎｋ，未知蛋白質、遺伝子＃Ａｔ２ｇ４７７７０，蛋白質 ｉｄ＝ＡＡＣ６３６３２．１，ｄｂ ｘｒｅｆ＝ＧＩ：３７３８２９０に対応する）は市販（Ｂｉｏｓｙｎｔｈｅｓｉｓ，Ｉｎｃ．Ｌｅｗｉｓｖｉｌｌｅ，ＴＸ）され、ＲＴ‐ＰＣＲ反応に使用される。たとえば、ＰＣＲ反応はそれぞれのプライマー ２５０ｎｇとゲノムＤＮＡ 約５００ｎｇを使用する。増幅反応の前に、成分を９５℃で２分間インキュベートする。遺伝子増幅反応は、９４℃で１分間、６５℃で１分間および７２℃で２分間を３０サイクル行い、その後７２℃で４分間延長して行う。
【００８５】
ＲＴ‐ＰＣＲでは、５′ＲＡＣＥ系（Ｌｉｆｅ Ｔｅｃｈｎｏｌｏｇｉｅｓ，Ｒｏｃｋｖｉｌｌｅ，ＭＤ）またはより簡単な逆転写酵素（ＲＴ）を使用して全長ｃＤＮＡクローンを同定する。プライマー３′ＥｃｏＰＢＲを使用して、取り扱い説明書に従い、２日目の植物から単離したポリ（Ａ⁺）ＲＮＡ１μｇ由来の第１鎖ｃＤＮＡを合成する。第１鎖ｃＤＮＡ合成物の１／５を、両プライマー、５′ＥｃｏＰＢＲおよび３′ＥｃｏＰＢＲによる遺伝子増幅反応における鋳型として使用する。増幅の前に成分を９５℃で２分間インキュベートする。遺伝子増幅反応は、９４℃で１分間、５８℃で１分間および７２℃で２分間を３０サイクル行い、その後７２℃で５分間を延長して行う。
【００８６】
ゲノムＤＮＡまたはｃＤＮＡフラグメントは、所望する結果に合わせて、センス（順方向）またはアンチセンス（逆方向）方向で植物形質転換ベクターにクローニングする。ベクターは構成的プロモータ、たとえばＣａＭＶ３５Ｓプロモータおよびノパリンシンターゼターミネータ、または本明細書に記載され、そして当技術分野で公知の他のプロモータを含むことができる。ベクターは改変されて生物的［Ｓｏｈａｌ ｅｔ ａｌ．、（１９９９）Ｐｌａｎｔ Ｍｏｌ．Ｂｉｏｌ．４１：７５−８７］または非生物的ストレス［Ｎｇａｉ ｅｔ ａｌ．（１９９７）Ｐｌａｎｔ Ｊ．１２：１０２１−１０３４；ｖａｎ Ｄｅｒ Ｋｒｏｌ ｅｔ ａｌ．，（１９９９）Ｐｌａｎｔ Ｐｈｙｓｉｏｌ．１２１：１１５３−１１６２；Ｋｕｃｈｏ ｅｔ ａｌ．（１９９９）Ｐｌａｎｔ Ｐｈｙｓｉｏｌ １２１：１３２９−１３３８］、および／またはホルモンまたは他のシグナル伝達分子［Ｃｈｅｎ ａｎｄ Ｓｉｎｇｈ（１９９９）Ｐｌａｎｔ Ｊ．１９：６６７−６７７；Ｌｕ ｅｔ ａｌ．（１９９８）Ｊ．Ｂｉｏｌ．Ｃｈｅｍ．２７３：１０１２０−１０１３１］；Ｌｅｕｂｎｅｒ−Ｍｅｔｚｇｅｒ ｅｔ ａｌ．（１９９８）Ｐｌａｎｔ Ｍｏｌ．Ｂｉｏｌ．３８：７８５−７９５］により誘導することができるプロモータを含んでいてもよい。クローニングされた構築物の配向は、制限エンドヌクレアーゼおよびＰＣＲ解析により確認する。
【００８７】
クローニングが完了すると、バイナリーベクターは、一部改変（すなわち、浸潤液への０．０２％（ｖ／ｖ）Ｓｉｌｗｅｔの添加）した減圧浸潤法［Ｂｅｃｈｔｏｌｄ，Ｎ．ａｎｄ Ｂｏｕｃｈｅｚ，Ｄ．（１９９５）Ｉｎ ｐｌａｎｔａ Ａｇｒｏｂａｃｔｅｒｉｕｍ−ｍｅｄｉａｔｅｄ ｔｒａｎｓｆｏｒｍａｔｉｏｎ ｏｆ ａｄｕｌｔ Ａｒａｂｉｄｏｐｓｉｓ ｔｈａｌｉａｎａ ｐｌａｎｔｓ ｂｙ ｖａｃｕｕｍ ｉｎｆｉｌｔｒａｔｉｏｎ．Ｉｎ Ｇｅｎｅ ｔｒａｎｓｆｅｒ ｔｏ Ｐｌａｎｔｓ．Ｉ．Ｐｏｔｒｙｋｕｓ ａｎｄ Ｇ．Ｓｐａｎｇｅｎｂｅｒｇ編．Ｓｐｒｉｎｇｅｒ−Ｖｅｒｌａｇ，Ｈｅｉｄｅｌｂｅｒｇ，ｐｐ１９−２３］を使用して、アグロバクテリウム‐ツメファシエンスの無害化系統、たとえばＥＨＡ１０５、続いてシロイヌナズナ（Ｗｓエコタイプ）に導入した。形質転換された植物から集めた種子を発芽させて、カナマイシン耐性に対して選択する。
【００８８】
本発明は図面および先の説明で詳細に説明され、記載されているが、同じものは特徴を説明するものであって、制限するものではないと見なすべきであり、好ましい態様だけが示され、そして記載されていること、および本発明の意図に含まれるすべての変更、および改変は保護されることが望ましいことを理解すべきである。さらに、本明細書に引用されたすべての参考文献は、当技術分野のレベルを表すものであって、参照としそのまま本明細書に援用する。
【００８９】
本発明の具体的な態様は特許請求の範囲においてとりわけ指摘されることになるが、発明自体、およびそれが作製され、使用される様式は本発明の一部を形成する添付の図面についての以下の説明を参照することにさらによく理解することができる。
【図面の簡単な説明】
【００９０】
【図１】植物ストレス反応において提案されたＧＡＢＡの役割を示した図である（ＧＡＢＡが環境ストレスシグナルの細胞バロメータおよびトランスジューサとして機能する仮説経路）。
【図２】ウキクサにおけるＧＡＢＡ‐媒介成長促進に対するシクロスポリンＡの作用を示すグラフであり、実施例１でより詳しく説明される。
【図３】ウキクサにおけるＧＡＢＡ‐媒介成長促進に対するスペルミンの作用を示すグラフであり、実施例１でより詳しく説明される。
【図４】ウキクサにおけるＧＡＢＡ‐媒介成長促進に対するキニーネの作用を示すグラフであり、実施例１でより詳しく説明される。
【図５】ウキクサにおけるＧＡＢＡ‐媒介成長促進に対するジアゼパムおよびＰＫ１１１９５（イソキノリンカルボキサミド）の作用を示すグラフであり、実施例１でより詳しく説明される。【Technical field】
[0001]
References for related patent applications
This patent application claims the benefit of US Provisional Application No. 60 / 306,819, filed June 20, 2001, which is incorporated herein by reference in its entirety.
[Background Art]
[0002]
Background of the Invention
The amino acid gamma-aminobutyric acid (GABA) is the major neurotransmitter in the mammalian central nervous system. Such neurotransmitters generally regulate the conductance of ions across the membrane of neurons, specifically the regulation of ion flux into cells. For example, GABA is believed to be an inhibitory neurotransmitter that inhibits synaptic transmission in both vertebrate and invertebrate nervous systems. As another example, glutamate is an excitatory neurotransmitter that depolarizes the postsynaptic membrane and promotes synaptic transmission. Both GABA and glutamate affect synaptic transmission by binding to their respective receptors, also known as ligand-gated ion channels.
[0003]
These ligand-gated ion channels are present in insect and animal neurons. Animal neurons have three general classes of GABA receptors, GABA_A, GABA_BAnd GABA_CExists. GABA receptors have been implicated in mediating anxiety, convulsions, cognitive function, addictive disorders, sleep disorders and other disorders of the central nervous system. GABA receptors are targets for many drugs acting on the central nervous system, including barbiturates and benzodiazepines, and are therefore of clinical importance. In addition, compounds that affect the function of insect GABA receptors are commercially useful as insecticides.
[0004]
GABA receptors have been found in the insect and animal kingdoms. Recently, proteins or other molecules that mediate the action of GABA and function as GABA receptors have been discovered in the plant kingdom (US patent application Ser. No. 09 / 517,438, Mar. 2, 2000). application).
[0005]
GABA has been shown to have positive beneficial effects on plants. For example, GABA has been shown to promote plant growth and productivity, as shown in Kinnersley US Pat. No. 5,439,873. In addition, such beneficial effects are promoted when GABA is applied with readily metabolized carbon sources such as succinic acid (US Pat. No. 5,604,177). Further, Kinnersley et al. As described in U.S. Patent No. 5,840,656, GABA has been found to enhance fertilizer efficiency when applied with glutamic acid.
[0006]
The mechanism of the beneficial effects of the above GABA in plants has not yet been identified. A better understanding of the mechanisms of GABA-mediated plant growth and productivity, and other mechanisms in which GABA is involved, could help another way to improve plant growth, productivity, and other beneficial effects Conceivable.
DISCLOSURE OF THE INVENTION
[0007]
Summary of the Invention
The present invention relates to the new discovery that plants respond to compounds known to act on animal mitochondrial GABA receptor proteins, and the related discovery that plants express receptor proteins responsive to these compounds. In this regard, the present invention provides nucleotide sequences that have been found in plants, which are believed to encode benzodiazepine or benzodiazepine-like receptors that have significant sensitivity to benzodiazepines. Based on the data presented herein, it is believed that such proteins function as modulators of GABA action, especially as ion channels, such as ligand-gated ion channels. In addition, such proteins are thought to be involved in plant stress-related physiological responses, and uptake of nucleic acid molecules encoding such proteins into plants is thought to promote the plant's ability to withstand stress. Accordingly, the present invention provides purified proteins, including recombinant proteins, nucleotide sequences encoding the proteins, and methods of using the nucleotide sequences and proteins.
[0008]
In one aspect of the invention, a method for transforming a plant is provided. In one aspect of the invention, a method includes introducing a nucleic acid molecule encoding a plant protein described herein into a plant cell.
[0009]
In a second aspect of the invention, a plant comprising providing a plant having an introduced nucleotide sequence encoding a plant protein described herein, and treating the plant with an effective amount of GABA. Is provided. In an alternative embodiment, the plants are treated with a composition comprising GABA and a GABA agonist, or are treated only with a GABA antagonist or GABA agonist. In another embodiment, the plant is treated with an animal benzodiazepine receptor agonist or antagonist, including an animal peripheral benzodiazepine receptor agonist or antagonist.
[0010]
In a third aspect of the invention, there is provided a method of regulating plant metabolism, comprising reducing the formation of a plant protein or RNA transcript, eg, mRNA, using antisense DNA or RNA. In one aspect, such a method involves introducing into a plant cell an antisense nucleic acid molecule having a nucleotide sequence complementary to the coding nucleotide sequence described herein, or a portion thereof. Alternatively, an antisense nucleic acid molecule includes a nucleotide sequence transcribed from a sequence described herein, preferably a nucleotide sequence that is complementary to an mRNA sequence. The antisense nucleotide sequence hybridizes to a nucleic acid containing either the plant template strand or the RNA transcript, and reduces the formation of the plant proteins described herein.
[0011]
In a fourth aspect of the present invention there is provided a method for identifying potential plant receptors, which comprises hybridizing a probe having a nucleotide sequence encoding a protein described herein, or a portion thereof, to a plant nucleic acid. Including doing.
[0012]
In a fifth aspect of the present invention there is provided a method for expressing a plant protein as described herein. In one embodiment, the method comprises introducing a nucleotide sequence encoding a plant receptor protein described herein into a host cell and culturing under conditions capable of expressing the receptor protein.
[0013]
In another aspect, there is provided an isolated nucleic acid molecule comprising a recombinant nucleic acid molecule, wherein such molecule comprises a nucleotide sequence encoding a plant protein described herein. Also provided are plant host cells and transgenic plants that include a nucleotide sequence encoding a plant protein described herein. Such molecules, plant cells and transgenic plants may further comprise a foreign promoter sequence operably linked to the 5 'end of the plant nucleotide sequences described herein.
[0014]
Description of the preferred embodiment
For a better understanding of the principles of the invention, preferred embodiments will now be described and specific terms will be used to describe them. However, without intending to thereby limit the scope of the invention, such modifications and further modifications of the invention, and such other applications of the principles of the invention described herein, are , Are contemplated as those of ordinary skill in the art.
[0015]
The present invention relates to the discovery that plants respond to compounds known to act on animal mitochondrial GABA receptor proteins, and the related discovery that plants express receptor proteins responsive to these compounds. The present invention further relates to the discovery of nucleotide sequences believed to encode plant benzodiazepine and / or benzodiazepine-like receptor proteins (hereinafter collectively referred to as "receptor proteins") in Arabidopsis thaliana. The invention also relates to nucleotide sequences that encode similar receptor proteins in other species and that exhibit similar function and sequence identity to the representative Arabidopsis sequences shown herein. Accordingly, the present invention provides an isolated nucleic acid molecule comprising a purified receptor protein and a nucleotide sequence encoding a plant receptor protein. Further provided are recombinant nucleic acid molecules, plant host cells and transgenic plants comprising a nucleotide sequence encoding a plant receptor protein. In other aspects of the invention, there are further provided methods of expressing the receptor proteins described herein, and methods of using nucleotide and amino acid sequences.
[0016]
In one aspect of the invention, a purified benzodiazepine or benzodiazepine-like receptor protein is provided. While not intending to limit the invention by any theory that the invention achieves its advantageous results, the plant receptor proteins described herein function as ligand-gated ion channel proteins in plants. Thus, it is thought to have the ability to regulate cellular ion influx and / or intracellular ion transport. Candidate ions whose influx can be regulated include anions such as chloride and cations such as calcium, sodium and potassium. For example, the receptor can release calcium ions from the intracellular store into the cytosol. According to this aspect of the invention, there is provided a substantially pure receptor protein. As used herein, “substantially pure” means that the receptor proteins are at least about 95% free of other proteins with which they are naturally present.
[0017]
In one aspect, the Arabidopsis receptor protein according to the present invention has the amino acid sequence set forth in SEQ ID NO: 2. Although the invention is described with reference to the Arabidopsis thaliana amino acid sequence, it is to be understood that the invention is not limited to the specific amino acid sequence set forth in SEQ ID NO: 2. One skilled in the art will recognize that throughout the course of mutation and / or evolution, polypeptides having different lengths and different components, eg, amino acid insertions, substitutions, deletions, etc., may occur, and such polypeptides may be produced. Is related or sufficiently similar to the sequences described herein and the advantageous functions described herein based on amino acid sequence homology. The terms "benzodiazepine receptor protein" and "benzodiazepine-like receptor protein" are generally used herein to describe proteins having the features described herein, one example of which is found in SEQ ID NO: 2. A polypeptide having the indicated amino acid sequence. In addition, variants of the polypeptides having structural features described herein and exhibiting function are included within this definition and within the scope of the invention.
[0018]
It is known that a wide variety of plants generally express and utilize homologous proteins, including the inserts, substitutions and / or deletions described above, and furthermore, Provides similar functionality. For example, an amino acid sequence isolated from another species may differ to some extent from the sequence set forth in SEQ ID NO: 2, although one of skill in the art would recognize it as a similar protein thought to have a similar function. It can be easily recognized. Amino acid sequences containing such mutations, having similar functions, and having a certain identity are included within the scope of the present invention. The present invention is not intended to be limited to any theory, such that the theory gives its favorable results, but the identity between the amino acid sequences required to maintain proper functioning is not a matter of three-dimensionality of the polypeptide. It is believed that in connection with maintaining the structure, the specific interactive sequence will be properly positioned and have the desired activity. It is contemplated that polypeptides containing these interacting sequences in an appropriate spatial environment will have sufficient activity even if other portions thereof are modified. In this regard, a variant of a protein described herein may include, for example, an amino acid that is conserved between various plant species, or a plant species that expresses a protein described herein. It may be considered functionally similar to the protein shown in SEQ ID NO: 2 if it contains unconserved amino acids present at certain positions.
[0019]
Another manner in which similarities can exist between two amino acid sequences is that certain amino acids of a group (eg, non-polar, uncharged, charged or basic) are the same. This is the case when it is replaced by another amino acid from the amino acid group. For example, the uncharged polar amino acid serine can generally be replaced with the uncharged polar amino acid threonine of the polypeptide without substantially altering the function of the polypeptide. Whether certain substitutions affect the function of the enzyme can be determined without undue experimentation by synthetic techniques and screening assays known in the art, including screening using the methods set forth in the Examples below. Can be checked using
[0020]
In one aspect, the present invention relates to amino acid sequences having at least about 60% identity to the amino acid sequence set forth in SEQ ID NO: 2 and exhibiting a function similar to the amino acid sequence set forth in SEQ ID NO: 2. provide. In another aspect, the present invention provides an amino acid sequence having at least about 70% identity to the amino acid sequence set forth in SEQ ID NO: 2 and exhibiting a function similar to the amino acid sequence set forth in SEQ ID NO: 2. A receptor protein having the formula: In another aspect, the present invention provides an amino acid sequence having at least about 80% identity to the amino acid sequence set forth in SEQ ID NO: 2 and exhibiting a function similar to the amino acid sequence set forth in SEQ ID NO: 2. A receptor protein having the formula: In another aspect, the present invention provides an amino acid sequence having at least about 90% identity to the amino acid sequence set forth in SEQ ID NO: 2 and exhibiting a function similar to the amino acid sequence set forth in SEQ ID NO: 2. A receptor protein having the formula:
[0021]
The percentage of identity can be determined, for example, from Oxford Molecular Group, Inc. (Beaverton, OR) MacVector computer program, version 6.0.1, and can be determined by comparing sequence information. Briefly, the MacVector program defines identity as the number of identical sequence symbols (ie, nucleotides or amino acids) divided by the total number of symbols in the shorter sequence of the two sequences. The program can be used to determine the percent identity over the entire length of the compared proteins. Preferred default parameters of the MacVector program include the following: For one pair of alignments: (1) matrix = BLOSUM30; (2) alignment speed-speed; (3) Ktuple = 1; (4) gap penalty = 1. Top diagonal = 5; window size = 5; for multiple alignments: matrix = BLOSUM series, open gap penalty = 10; enlarged gap penalty = 0.1, delayed divergence = 40%; protein gap parameter: gap isolation distance = 8; Residue specific penalty = yes or on; hydrophilic residue = GPSNDQEKR.
[0022]
In another aspect of the invention, there is provided an isolated nucleic acid molecule encoding a protein described herein. In one aspect, the invention provides the nucleotide sequence set forth in SEQ ID NO: 1, originally isolated from Arabidopsis thaliana. In addition, it is to be understood that sequences complementary to the specific sequences set forth therein are also encompassed by the present invention. In one aspect of the invention, the nucleic acid comprises a nucleotide sequence encoding a protein having an amino acid sequence having at least about 60% identity to the amino acid sequence set forth in SEQ ID NO: 2, and set forth in SEQ ID NO: 2. An isolated nucleic acid molecule that exhibits a function similar to an amino acid sequence is provided. In another aspect, the invention has a nucleotide sequence that encodes a protein having an amino acid sequence having at least about 70% identity to the amino acid sequence set forth in SEQ ID NO: 2, and is set forth in SEQ ID NO: 2. The present invention provides an isolated nucleic acid molecule exhibiting a function similar to the amino acid sequence of the present invention. In another aspect, the invention has a nucleotide sequence that encodes a protein having an amino acid sequence having at least about 80% identity to the amino acid sequence set forth in SEQ ID NO: 2, and the sequence set forth in SEQ ID NO: 2. The present invention provides an isolated nucleic acid molecule exhibiting a function similar to the amino acid sequence of the present invention. In another aspect, the invention has a nucleotide sequence that encodes a protein having an amino acid sequence having at least about 90% identity to the amino acid sequence set forth in SEQ ID NO: 2, and is set forth in SEQ ID NO: 2. The present invention provides an isolated nucleic acid molecule exhibiting a function similar to the amino acid sequence.
[0023]
The present invention is not limited to these representative nucleotide sequences, but includes sequences having substantial similarity thereto and variants of the plant receptor proteins described hereinbefore and further described below. It is intended to encompass sequences encoding
[0024]
The term "isolated nucleic acid" as used herein is intended to describe a nucleic acid that is not in its natural environment. For example, the term refers to nucleic acids separated from other contaminants that naturally accompany it, such as proteins, lipids, and other nucleic acid sequences. Such terms include nucleic acids that have been removed or purified from their naturally occurring environment or from a clonal library, and further include recombinant or cloned nucleic acid isolates and chemically synthesized nucleic acids. .
[0025]
The term "nucleotide sequence" as used herein refers to a natural or synthetic linear and continuous array of nucleotides and / or nucleosides and is intended to include deoxyribonucleic acids, ribonucleic acids, and derivatives thereof. The terms "encoding" and "coding" refer to the process by which a nucleotide sequence provides information to a cell by mechanisms of transcription and translation, whereby a series of amino acids is associated with a specific amino acid sequence, and the functional polypeptide For example, an active enzyme or other protein having a specific function can be produced. The process of encoding a particular amino acid sequence may include a DNA sequence having one or more base changes (ie, insertions, deletions, substitutions) that do not cause a change in the encoded amino acid, or Can change, but may include base changes that do not destroy the functional properties of the polypeptide encoded by the DNA sequence.
[0026]
Accordingly, it is to be understood that the present invention includes more than the specific exemplary nucleotide sequences set forth in SEQ ID NO: 1. For example, nucleic acid sequences encoding the variant amino acid sequences described above are within the scope of the invention. Sequence alterations such as deletions, insertions, or substitutions that result in "silent" changes that do not substantially affect the functional properties of the resulting polypeptide molecule are clearly contemplated by the present invention. For example, it should be understood that modifications of the nucleotide sequence that reflect the degeneracy of the genetic code or that result in the production of chemically equivalent amino acids at certain sites are contemplated. Thus, the codon of the amino acid alanine, a hydrophobic amino acid, is replaced by another less hydrophobic residue, such as glycine, or a more hydrophobic residue, such as a codon encoding valine, leucine, or isoleucine. Can be. Similarly, if such altered nucleotide sequence is expected to produce a bioequivalent product, the conversion of other residues to one negatively charged residue, such as a substitution of glutamic acid for aspartic acid, is considered. Modifications, such as substitutions or substitutions of other residues with one positively charged residue, such as substitution of arginine for lysine, are also contemplated by the present invention.
[0027]
Also, nucleotide changes that alter the N-terminal and C-terminal portions of the encoded polypeptide molecule typically do not appear to alter the activity of the polypeptide. In such cases, it is desirable to mutate the sequence and, in effect, examine the effect of the alteration on the biological activity of the polypeptide. Each proposed modification is well within the routine routine of the art.
[0028]
In one preferred aspect, the present invention provides nucleotide sequences having substantial similarity to the entire sequence set forth in SEQ ID NO: 1, and variants described herein. The term "substantial similarity" is used herein with reference to nucleotide sequences, in which such a nucleotide sequence is sufficiently similar to a reference nucleotide sequence that it will hybridize under moderately stringent conditions. To have This method for determining similarity is known in the art to which the present invention belongs. Briefly, moderately stringent conditions are described in Sambrook et al. , Molecular Cloning: A Laboratory Manual, 2nd ed., 1 vol. 101-104, described in Cold Spring Harbor Laboratory Press (1989), 5X SSC (sodium chloride / sodium citrate solution), 0.5% sodium dodecyl sulfate (SDS), 1.0 mM ethylenediaminetetraacetic acid (EDTA) (pH 8) 0.0) pre-wash solution and hybridization and the use of wash conditions of 55 ° C, 5X SSC. Another requirement of the polynucleotide of the present invention is that it must encode a polypeptide having a function similar to the plant proteins described herein.
[0029]
In yet another aspect, there is provided a nucleotide sequence having a selected percentage of identity to a particular region of the nucleotide sequence set forth in SEQ ID NO: 1. In one aspect of the invention, there is provided a nucleotide sequence having at least about 50% identity to a nucleotide sequence of substantial length within the nucleotides set forth in SEQ ID NO: 1. In another aspect, the invention provides a nucleotide sequence having at least about 60% identity to a nucleotide sequence of substantial length within the nucleotides set forth in SEQ ID NO: 1. In another aspect, the invention provides a nucleotide sequence having at least about 70% identity to a nucleotide sequence of substantial length within the nucleotides set forth in SEQ ID NO: 1. In another aspect, the invention provides a nucleotide sequence having at least about 80% identity to a nucleotide sequence of substantial length within the nucleotides set forth in SEQ ID NO: 1. In another aspect, the invention provides a nucleotide sequence having at least about 90% identity to a nucleotide sequence of substantial length within the nucleotides set forth in SEQ ID NO: 1.
[0030]
In one aspect, "substantial length" refers to a length of at least about 50 nucleotides. In another aspect, the substantial length is at least about 100 nucleotides in length. In another aspect, the substantial length is at least about 200 nucleotides in length. In another aspect, the substantial length is at least about 300 nucleotides in length. In another aspect, the substantial length is at least about 400 nucleotides in length. In another aspect, the substantial length is at least about 500 nucleotides in length. In another embodiment, the substantial length is the entire sequence set forth in SEQ ID NO: 1.
[0031]
Percent identity can be determined, for example, by comparing sequence information using the MacVector program, described above for amino acid identity. Preferred default parameters include: (1) a pair of alignment parameters: (a) Ktuple = 1; (b) gap penalty = 1; (c) window size = 4; and (2) multiple alignment. Parameters: (a) open gap penalty = 10; (b) enlarged gap penalty = 5, (c) delay divergence = 40%; and (d) transition = weighted. Another requirement for a nucleotide sequence according to the present invention is that it encodes a protein that functions as described herein.
[0032]
Suitable DNA sequences according to the present invention can be obtained by cloning techniques using Arabidopsis or other species of cDNA or genomic libraries, which are either commercially available or known in the art. Can be constructed using standard methods. Suitable nucleotide sequences include those selected according to the present invention as probes or primers, such as those set forth in SEQ ID NO: 1; nucleotide sequences having substantial similarity thereto, or portions thereof. Can be isolated from DNA libraries obtained from various species by nucleic acid hybridization or polymerase chain reaction (PCR) methods. In a preferred embodiment of the present invention, the nucleotide sequence provided herein is a cDNA sequence.
[0033]
Alternatively, appropriate sequences can be made by techniques known in the art. For example, a nucleic acid sequence encoding a plant protein described herein can be constructed using recombinant enzymes, such as nucleic acid cleavage or splicing, using restriction enzymes and DNA ligase. In addition, nucleic acid sequences can be constructed using chemical synthesis, eg, solid phase phosphoramidite technology, or PCR. PCR can also be used to increase the amount of nucleic acid produced. Furthermore, if the particular nucleic acid sequence is of a length that renders full-length chemical synthesis impossible, the sequence may be divided into small portions, synthesized by methods known in the art, and ligated together. The desired complete arrangement can be formed.
[0034]
In another aspect of the present invention, there is provided a recombinant nucleic acid molecule, or a recombinant vector. In one aspect, there is provided a nucleic acid molecule comprising a nucleotide sequence described herein. The protein encoded by the nucleotide sequence has the amino acid sequence set forth in SEQ ID NO: 2, or a variant thereof as described above.
[0035]
The various vectors used in the present invention are known. For example, various plasmids and phages that are ideally suited for use in the present invention are known and include λZap and pBluescript. In a preferred embodiment, the vector may be a T-DNA vector. Representative T-DNA vector systems are described in the following publications: An et al. , (1986) EMBO J .; 4: 277; Herrera-Estrella et al. , (1983) EMBO J .; 2: 987; Herrera-Estrella et al. , (1985) in Plant Genetic Engineering, New York: Cambridge University Press, p. 63.
[0036]
In one aspect, the desired recombinant vector has a DNA linker sequence linked to the 5 'and 3' ends of the desired nucleotide insert, as is known in the art, to unambiguously recognize the linker sequence and the sequences present in the desired vector. By cleaving the insert with a restriction enzyme, cleaving the vector with the same restriction enzyme, mixing the cleaved vector with the cleaved insert, and incorporating the insert into the vector using DNA ligase. Can be.
[0037]
The vector can include other nucleotide sequences, eg, sequences encoding a selectable marker, including markers for antibiotic resistance or color selection. The vector also preferably contains a promoter nucleotide sequence. The desired nucleic acid insert is preferably operably linked to a promoter. A nucleic acid is “operably linked” to another nucleic acid sequence, such as a promoter sequence, when it is placed into a specific functional relationship with another nucleic acid sequence. A functional association between a promoter and a desired nucleic acid insert generally includes the flanking nucleic acid and promoter sequence such that transcription of the nucleic acid sequence is promoted. The nature of the linkage between the two nucleic acid sequences may result in: (1) introducing a frameshift mutation; (2) interfering with the ability of the promoter region sequence to transcribe the desired nucleotide sequence; or (3) transcription by the promoter sequence region. The two nucleic acid sequences are said to be further operably linked if they do not interfere with the ability of the desired nucleotide sequence to be performed. Typically, the promoter element is generally upstream (ie, at the 5 'end) of the nucleic acid insert coding sequence.
[0038]
A variety of promoters are known in the art, including cell-specific promoters, inducible promoters, and constitutive promoters. In plant cells, promoters for transcription can be used. Promoters may be of viral, bacterial or eukaryotic origin, including those of plant and plant virus origin. For example, in certain preferred embodiments, the promoter may be of viral origin, including a cauliflower mosaic virus promoter (CaMV), such as CaMV35S or 19S, a sesame clover mosaic virus promoter (FMV35S), or a tobacco mosaic virus (TMV). Coat protein promoter. Further, the promoter may be, for example, the promoter of the small subunit of ribulose-1,3-bisphosphate carboxylase. Promoters of bacterial origin include Herrera-Estrella et al. Octopain synthase promoter, nopaline synthase promoter and other promoters from natural Ti plasmids as described in J., Nature, 303: 209-213 (1983).
[0039]
In addition, the promoter may be responsive to various forms of environmental stress or other stimuli. For example, promoters may be abiotic stresses such as trauma, cold, dry, UV-B [van Der Krol et al. (1999) Plant Physiol. 121: 1153-1162], heat shock [Shinmyo et al. (1998) Biotechnol. Bioeng. 58: 329-332] or other heat, drought, or water stress.
[0040]
In addition, promoters may be those induced by biological stress, including pathogen stress, such as viruses [Sohal et al. (1999) Plant Mol. Biol. 41: 75-87] or a fungus [Eulgerm (1999) EMBO. J. 18: 4689-4699], as part of the plant defense pathway [Lebel (1998) Plant J. et al. 16: 223-233], or other environmental signals such as light [Ngai et al. (1997) Plant J. et al. 12: 1021-1034; Sohal et al. (1999) Plant Mol. Bio. 41: 75-87], carbon dioxide [Kucho et al. (1999) Plant Physiol 121: 1329-1338], hormones or other signaling molecules such as auxin, hydrogen peroxide and salicylic acid [Chen and Singh (1999) Plant J. et al. 19: 667-677], sugars and gibberellins [Lu et al. (1998) J. Am. Biol. Chem. 273: 10120-10131] or abscisic acid and ethylene [Leubner-Metzger et al. (1998) Plant Mol. Biol. 38: 785-795].
[0041]
In addition, they may be selected such that the promoter requires activation by other elements known in the art, such that the production of the protein encoded by the nucleic acid sequence insert is regulated as desired. Can be. In one aspect, the promoter is a foreign promoter. As used herein, an "exogenous promoter" is described to mean a promoter other than the native or natural promoter that promotes the transcription of a piece of DNA.
[0042]
In addition, the vector may include other regulatory elements, such as enhancer sequences, which cooperate with a promoter to effect transcription of the nucleic acid insert coding sequence. By the term "enhancer" is meant a nucleotide sequence element capable of stimulating promoter activity in a cell, such as a plant host cell. The vector may further include 3 'regulatory sequence elements known in the art, for example, to increase the stability of the transcribed RNA.
[0043]
In addition, the vector may include another nucleotide sequence insert, which insert encodes a peptide or polypeptide used as a tag to assist in purifying the desired protein encoded by the desired nucleotide sequence. Or encode another functional protein. With respect to inclusion of a tag, additional nucleotide sequences can be placed in the vector to obtain a fusion, or chimeric protein. For example, a protein described herein having a linker amino acid at the C-terminus and attached to another protein that acts as a tag can be produced as is known in the art. After purification procedures known to those skilled in the art, the additional amino acid sequence is cleaved by a suitable enzyme. Thereafter, the protein can be isolated from other proteins, or fragments thereof, by methods known in the art.
[0044]
In another embodiment, the vector comprises a second nucleotide sequence, wherein said sequence is another functional protein, such as the plant described in our co-pending US patent application Ser. No. 10 / 006,852. Encodes GAD enzyme. Alternatively, the plant is transformed according to the present invention with two vectors, for example, one containing a DNA construct for expression of the GAD enzyme, and one for expression of the plant receptor protein described herein. Can be. It is believed that overexpression of GAD enzymes and receptor proteins in certain plants will result in plants with superior characteristics, for example, strong stress resistance.
[0045]
The recombinant vector of the present invention can be used to transform a host cell. Accordingly, there is provided a method of transforming a cell or plant, comprising introducing a nucleic acid molecule having the nucleotide sequence of the present invention into a plant cell. Various methods for transforming cells or plants are known in the art and are described, for example, in Maniatis et al. , Molecular Cloning: A Laboratory Manual, Cold Springs Laboratory, Cold Springs Harbor, New York (1982) and Current Protocols in Molecular Biology, Molecular Biology, Molecular Biology, Molecular Biology, Molecular Biology, Molecular Biology, Molecular Biology, Molecular Biology, Molecular Biology, Molecular Biology, Molecular Biology, Molecular Biology, Molecular Biology, Molecular Biology, Molecular Biology, Molecular Biology, Molecular Biology, Molecular Biology, Molecular Biology, Molecular Biology, Molecular Biology, Molecular Biology, Molecular Biology, Molecular Biology, Molecular Biology, Molecular Biology, Molecular Biology, Molecular Biology, Molecular Biology, Molecular Biology, Molecular Biology, Molecular Biology, Molecular Biology, Molecular Biology. Ed. (1998). Plant gene transfer techniques can also be found in references, including: Fromm et al. , (1985) Proc. Natl. Acad. Sci. ScL USA, 82: 5824-5828 (lipofection); Crossway et al. , (1986) Mol. Gen. Genet. 202: 179 (microinjection); Hoykaas-Van Slogtern et al. , (1984) Nature 311: 763-764) (T-DNA mediated transformation of monocotyledonous plants); Rogers et al. , (1986) Methods Enzymol. 118: 627-641 (T-DNA mediated transformation of dicotyledonous plants); Bevan et al. , (1982) Ann. Rev .. Genet. 16: 357-384) (T-DNA mediated transformation of dicotyledonous plants); Klein et al. , (1988) Proc. Natl. Acad. Sci USA 85: 4305-4309 (particle gun method); and Fromm et al. , Nature (1986) 319: 791-793 (electroporation). In a suitable vector, the introduced polynucleotide is advantageously integrated into the plant genome, but can remain episomal in other aspects of the invention. Once the desired nucleic acid has been introduced into the host cell or host plant, the host cell will express the protein. Thus, in a further aspect of the present invention there is provided a host cell comprising a recombinant vector of the invention as described above.
[0046]
A variety of host cells can be used in the present invention, including prokaryotic and eukaryotic host cells. Preferred host cells are eukaryotic cells, and more preferably are plant cells derived from, for example, plants such as the following: monocotyledonous plants, such as duckweed, corn, turf (eg, ryegrass, arthrush, strawberry rush, oxwort). Dicotyledonous plants, such as cereals such as lettuce and wheat, cross plants (eg, rape, radish and cabbage), solanaceous plants (eg, peppers, potatoes and tomatoes), and legumes (eg, soybeans and kidney beans). In another aspect of the invention, the host cells can be cultured to produce transgenic plants as known in the art. A transformed plant is transformed, for example, from a host plant-derived cell, tissue or organ with a nucleic acid molecule of the invention; selecting a transformed cell, cell callus, somatic embryo, or seed containing the nucleic acid molecule; Can be produced by regenerating whole plants from transformed cells, cell calli, somatic embryos, or seeds; and selecting regenerated whole plants that express nucleic acid sequences.
[0047]
In another aspect of the invention, a method is provided for identifying a plant protein, for example, a protein that is considered to be a benzodiazepine or benzodiazepine-like receptor. In these methods, the nucleotide sequences described above, or portions thereof, are used as probes to discover another similar nucleotide sequence that can encode another benzodiazepine or benzodiazepine-like receptor. General methods for screening selected nucleotide sequences in DNA or RNA samples are known in the art. For example, DNA can be isolated from selected plants, treated with various restriction enzymes, and analyzed by Southern blotting using a radioactive or fluorescently labeled probe of interest. RNA fragments can be similarly analyzed by Northern blotting. Alternatively, commercially available cDNA or genomic libraries can be screened.
[0048]
In one aspect, the nucleic acid molecule used as a probe has a nucleotide sequence having at least about 60% identity to a nucleotide sequence of about 25 to about 100 nucleotides in length within the nucleotide sequence set forth in SEQ ID NO: 1. . In another aspect, the nucleic acid molecule used as a probe comprises a nucleotide sequence having at least about 60% identity to a nucleotide sequence from about 25 to about 400 nucleotides in length within the nucleotide sequence set forth in SEQ ID NO: 1. Have. In another aspect, the nucleic acid molecule used as a probe comprises a nucleotide sequence having at least about 60% identity to a nucleotide sequence of about 25 to about 500 nucleotides in length within the nucleotide sequence set forth in SEQ ID NO: 1. Have. In another aspect, the nucleic acid molecule used as a probe has a nucleotide sequence that has at least about 60% identity to the entire length of the nucleotide set forth in SEQ ID NO: 1. In another embodiment, the probe has a nucleotide sequence that is at least about 70% identical to a nucleotide of the length indicated immediately above. In another embodiment, the probe has a nucleotide sequence that is at least about 80% identical to a nucleotide of the length indicated immediately above. In another aspect, the probe has a nucleotide sequence that is at least about 90% identical to nucleotides of the length indicated immediately above. Probes include, for example, polynucleotide kinase and³²The 5 'end can be radiolabeled with P and hybridized to the isolated nucleic acid fragment.
[0049]
In another aspect of the present invention, there is provided a method of treating a plant. In one aspect, the method includes providing a plant having the introduced nucleic acid molecule described herein and expressing the encoded receptor protein, and treating the plant with an effective amount of GABA. It is believed that treating such plants advantageously provides other beneficial results, including stimulating plant growth and reducing the effects of plant stress.
[0050]
In one aspect, the transgenic plants are made as described above and treated with an effective amount of GABA. As used herein, “effective amount” refers to an amount of GABA that provides one or more benefits to a plant, such as, for example, stimulating plant growth and / or reducing plant stress. The amount may vary depending on a variety of factors including, for example, the specific benefits provided to the plant, the amount of expression of the introduced nucleotide sequence, the type of plant, the number of plants to be treated and the environmental conditions. . In one aspect, the plant is about 1 ppm to about 24,000 ppm [about 0.013 oz / acre (oz / A) to about 20 lbs / A] [about 0.93 g / ha (g / ha) to about 22 kg / ha] GABA Is processed. In another embodiment, the plants are treated with about 1 ppm to about 12,000 ppm [about 0.013 oz / A to about 10 lbs / A] [about 0.93 g / ha to about 11 kg / ha] GABA. In another embodiment, the plant is treated with about 1 ppm to about 7,500 ppm [about 0.013 oz / A to about 6.3 lbs / A] [about 0.93 g / ha to about 7.1 kg / ha] GABA. In another aspect, the plant is treated with about 1 ppm to about 5.000 ppm [about 0.013 oz / A to about 4.2 lbs / A] [about 0.93 g / ha to about 4.8 kg / ha] GABA. For plant growth stimulation, concentrations from about 1 ppm to about 5,000 ppm can be conveniently used, as described in Kinnersley US Pat. No. 5,439,873. If a reduction in plant stress is desired, a GABA concentration of about 1 ppm to about 2500 ppm [about 0.013 oz / A to about 2.1 lbs / A] [about 0.93 g / ha to about 2.4 kg / ha] may be used. Can be used conveniently. In one aspect of the invention, about 150-600 ppm [about 1/8 lb / A to about 1/2 lb / A] [about 0.14 kg / ha to about 0.56 kg / ha] is used. All amounts in ppm are weight / volume (g / ml). In addition, the percentages of application in parentheses above are sought for processes that use a standard amount of 100 gallons of a particular solution spread over one acre.
[0051]
In yet another aspect, in addition to being treated with GABA, the plant may be treated with a composition comprising GABA and a GABA agonist. For example, plants can be baclofen and GABA agonists known in the art, such as cis-4-aminopent-2-enoic acid (CACA), imidazole-4-acetic acid (IAA) and 4,5,6,7, -tetrahydroiso It may be treated with xazolo [5,4-c] pyridin-3-ol (THIP). Also, plants can be treated with only a GABA antagonist, such as picrotoxin or bicuculline, or only a GABA agonist, to regulate plant metabolism as desired. Plants can also be treated only with benzodiazepine receptor agonists or antagonists, such as animal peripheral benzodiazepine receptors. Such compounds include quinine and spermine, and other benzodiazepine receptor antagonists and agonists described herein.
[0052]
GABA, GABA agonists or antagonists and other agonists and antagonists described herein generally apply to plant leaves, but may also be applied as soil drench. Furthermore, when plants are hydroponically grown, the compounds and compositions can be applied to the aqueous solution in which the plants are grown. The composition is further applied, preferably by spraying. In addition, the compounds and compositions can be applied as a seed treatment.
[0053]
GABA, GABA agonists or antagonists and other agonists and antagonists described herein are preferably combined with a carrier medium known in the art. The compounds and compositions can be combined with, for example, water, such as tap water, or distilled water to which selected minerals have been added. Alternatively, the compositions of the present invention can be applied as a solid. In such a form, the solid is preferably applied to the soil. In addition, the compositions can include agricultural additives or formulation auxiliaries known to those skilled in the art. Such additives or auxiliaries ensure that the composition is well dispersed in the sprayer, adheres or penetrates the plant surface, especially leaves or other foliar surfaces, and provides other benefits to the plant Can be used for For example, surfactants, dispersants, wetting agents, and binders are used to disperse the compound or composition in the sprayers described herein and adhere and / or penetrate the compound or composition to plant surfaces. Can be done.
[0054]
Further, a method for regulating plant metabolism is provided by the present invention. Regulation of plant metabolism includes having a positive or negative impact on nutrient utilization such as nitrogen assimilation, plant growth, plant productivity, and the plant's resistance to the effects of plant stress. For example, in one aspect, a method of the invention that can negatively affect plant productivity includes introducing an antisense nucleotide sequence having a sequence complementary to a coding nucleotide sequence described herein into a plant cell. It is mentioned.
[0055]
Thus, the present invention also provides methods for engineering genes involved in plant receptor protein production, and is therefore a valuable tool for further study of cell stress and / or growth processes. For example, manipulation of plant receptor protein genes can provide quantitative information on the role of GABA-related processes on metabolic flux, nutrient utilization and storage, cell differentiation, growth, senescence, and signal transduction. Further, such operations provide a way to increase crop productivity by increasing the crop's resistance to biological and abiotic stresses. Crop quality and yield are improved by increasing the resistance to various environmental stresses, including diseases, that reduce the photosynthesis and nitrogen efficiency of the crop plant and reduce yield.
[0056]
In one aspect, the invention provides an antisense nucleotide sequence that is complementary to a nucleotide sequence that has at least about 50% identity to a particular nucleotide within the nucleotide sequence set forth in SEQ ID NO: 1. In another aspect, the invention provides an antisense nucleotide sequence that is complementary to a nucleotide sequence that has at least about 60% identity to a particular nucleotide within the nucleotide sequence set forth in SEQ ID NO: 1. In another aspect, the invention provides an antisense nucleotide sequence that is complementary to a nucleotide sequence that has at least about 70% identity to a particular nucleotide within the nucleotide sequence set forth in SEQ ID NO: 1. In another aspect, the invention provides an antisense nucleotide sequence complementary to a nucleotide sequence having at least about 80% identity to a particular nucleotide within the nucleotide sequence set forth in SEQ ID NO: 1. In another aspect, the invention provides an antisense nucleotide sequence that is complementary to a nucleotide sequence that has at least about 90% identity to a particular nucleotide within the nucleotide sequence set forth in SEQ ID NO: 1.
[0057]
In one aspect, the antisense nucleotides are from about 30 to about 100 nucleotides in length. In another aspect, the antisense nucleotides are from about 30 to about 200 nucleotides in length. In another aspect, the antisense nucleotides are from about 30 to about 300 nucleotides in length. In another aspect, the antisense nucleotides are from about 30 to about 400 nucleotides in length. In another aspect, the antisense nucleotide sequence is the same length as the entire nucleotide sequence shown in SEQ ID NO: 1. The antisense nucleotide sequence is capable of hybridizing to a template strand that serves as the strand from which RNA is produced, thereby inhibiting transcription. Alternatively, the antisense nucleotide sequence is complementary to, and thus hybridizable to, an RNA sequence, such as an mRNA sequence transcribed from a nucleotide sequence described herein, so that the mRNA to express the encoded protein is Sequence transcription will be inhibited. The antisense nucleotide sequence can be either DNA or RNA. Preferred antisense oligonucleotides are complementary to the coding region of a particular polynucleotide, but moreover sequences can bind to selected sequences in non-coding regions. In a further preferred embodiment of the invention, the antisense oligonucleotide binds to a nucleotide adjacent to the ATG start codon.
[0058]
In another aspect of the present invention, there is provided a method of modulating plant metabolism by in vivo mutagenesis of a gene present in a plant genome encoding the same, in order to alter the activity of a plant receptor protein described herein. And provide the desired positive or negative results described above. Plants can be mutated by methods known to those skilled in the art, for example, chemical methods and DNA insert activation tag mutagenesis.
[0059]
In another aspect of the present invention, there is provided a method of modifying a receptor activity of a plant. In one aspect of the invention, a method comprises introducing a nucleic acid molecule having a nucleotide sequence encoding a plant protein described herein into a plant cell.
[0060]
In still another aspect of the present invention, there is provided a method for expressing a plant protein which is thought to function as the benzodiazepine receptor. In one aspect, the method comprises providing a nucleotide sequence as described above, or a variant thereof, encoding a protein described herein, and introducing the nucleotide sequence into a host cell as described above. . The desired nucleotide sequence can be conveniently introduced into the vector to form a recombinant vector. The recombinant vector can then be introduced into a host cell according to procedures known in the art. Such host cells are then cultured under conditions known to those skilled in the art and effective for expression of the plant protein. The protein can then be purified using conventional techniques.
[0061]
A variety of target plants are contemplated according to the present invention. In one aspect, the target plant is selected from the group consisting of the following plants: duckweed, rice, wheat, barley, rye, corn, sycamore, strawberry, grasshopper, bollwort, rapeseed, potato, carrot, sweet potato, soy beans, peas. , Chicory, lettuce, cabbage, cauliflower, broccoli, turnip, radish, spinach, asparagus, onion, garlic, eggplant, capsicum, celery, squash, pumpkin, zucchini, cucumber, apple, pear, quince, melon, plum, cherry Peaches, nectarines, apricots, strawberries, grapes, raspberries, blackberries, pineapples, avocados, papayas, mangos, bananas, soybeans, kidney beans, tobacco, tomatoes, peppers, sorghum and sugarcane.
[0062]
Any experiments, examples, or experimental results provided herein are illustrative of the present invention and should not be deemed to limit or limit the scope of the present invention. In addition, any theory, mechanism of action, or discovery described in the present invention is for further understanding of the present invention and should not be construed as limiting the present invention in any way to such theory, mechanism or discovery. Not intended to be limiting. All publications, patents, and patent applications cited herein are each and every publication, patent, or patent application specifically and individually as if it were required by reference to be incorporated by reference. And is hereby incorporated by reference and as it is herein. While the invention has been described and described in detail in the drawings and foregoing description, the same should be considered as describing features and not as limiting, and only selected embodiments are illustrated. All changes, equivalents, and modifications that come within the spirit of the invention as described and described herein and as described herein or as set forth in the following claims are protected. Should be understood to be desirable.
[0063]
Reference will now be made to specific examples illustrating the invention described above. It should be understood that the examples are provided to illustrate preferred embodiments and are not intended to limit the scope of the invention.
Embodiment 1
[0064]
Effects of agonists and antagonists of animal mitochondrial benzodiazepine receptor on GABA-mediated growth promotion of duckweed
Benzodiazepine receptors are sensitive to the agonist diazepam and the antagonist PK11195 (isoquinolinecarboxamide), spermine, quinine and cyclosporin A.
[0065]
The medium was Solu-Spray 20-20-20 fertilizer dissolved in tap water at 1 g / l as described in Kinnersley US Patent No. 5,439,873, except that the pH was adjusted to 5.5. Duckweed (Lemna Minor L) were grown according to the general procedure described by Kinnersley (US Pat. No. 4,813,997). Duckweed were separately treated with the indicated concentrations of GABA and cyclosporin A, spermine, quinine, diazepam or PK11195.
[0066]
As seen in FIGS. 2, 3 and 4, when treated separately with cyclosporin A, spermine, or quinine in the presence of GABA in the medium, an inhibitory effect on growth was seen. Cyclosporin A is an immunosuppressant and has been shown to be the most potent pharmacological inhibitor of animal mitochondrial PTP. The inhibitory activity of cyclosporin is due to the binding of the inner mitochondrial membrane to mitochondrial cyclophilin. In duckweed experiments, 3 μM cyclosporin A significantly inhibited plant growth in cultures containing 10 mM GABA. Compared to the respective controls, the inhibition of GABA-mediated growth by cyclosporin A (FIG. 2), spermine (FIG. 3) and quinine (FIG. 4) was paradoxically the strongest at the highest levels of GABA. This is most clearly seen in FIG. 3, where the dry weight of the culture with 10 mM GABA and 150 μM spermine was significantly less than the culture without GABA and with 150 μM spermine. Addition of 3 μM of the benzodiazepine diazepam to the cultures promoted GABA-mediated growth (FIG. 5). Growth promotion was significant at P <0.05. Also, the effect of diazepam on GABA activity in plants is further evidence of similarity of GABA receptor structure in animals and plants.
[0067]
Furthermore, when duckweed was treated with PK11195 in the absence of GABA, an inhibitory effect on growth was observed as shown in Table 1 below. PK11195 is a characteristic ligand of the peripheral benzodiazepine receptor and is associated with PTP in animal mitochondria. PK11195 inhibited GABA-mediated growth reaction at 50 μM (FIG. 5).
[0068]
[Table 1]

[0069]
The data in Table 1 suggest that when low levels of GABA, such as endogenous GABA levels, are present in the plant, high concentrations of PK11195 are required to confirm action.
As shown in FIG. 5, when the duckweed grown as described above was independently treated with diazepam, a growth promoting effect was observed in the presence of GABA in the medium. When duckweed was independently treated with PK11195, growth inhibition was observed in the presence of GABA in the medium.
[0070]
Earlier studies reported the effects of animal GABA receptor antagonists on duckweed growth; however, the inhibitors of GABA bioactivity reported in Table 2 below were higher than the GABA receptor antagonists used in earlier studies. The activity was up to 1000-fold. This suggests that they act on different classes of GABA receptors, and that, like animals, plants also have diverse GABA receptors.
[0071]
[Table 2]

[0072]
Experiments with drugs that promote or inhibit the activity of benzodiazepine receptors in animals have shown similar results in plants, and together with the above results provide evidence for benzodiazepines or benzodiazepine-like receptors in plants I do.
Embodiment 2
[0073]
Isolation of full-length cDNA and genomic DNA
Protocol
Arabidopsis thaliana (L.) Heynh. Seeds of the ecotype Columbia (Col-0) can be obtained from the Arabidopsis Biological Resource Center (Ohio State University, Columbia, OH). Arabidopsis seedlings were grown on a rotary shaker (150 rpm) in an MS medium [Murashige and Skook, Physiol. Plant 15: 485 (1962)]. Day 2 seedlings are harvested for total RNA isolation. Total RNA is from Turano, F.C. J. et al. (1992) Plant Physiol. Isolate as described at 100: 374. Synthesized primer
[0074]
Embedded image

[0075]
and
[0076]
Embedded image

[0077]
(SEQ ID NOs: 3 and 4, respectively) (GenBank, unknown protein, gene # At2g47770, protein id = AAC63632.1, db xref = GI: 3738290) is commercially available (Biosynthesis, Inc. Lewisville, Tex.) and is used in RT-PCR reactions. In RT-PCR, a full length cDNA clone is identified using the 5 'RACE system (Life Technologies, Rockville, MD). Poly (A) isolated from day 2 plants using primer 3'EcoPBR according to the manufacturer's instructions⁺1) First strand cDNA from 1 μg of RNA is synthesized. One fifth of the first strand cDNA synthesis is used as a template in a gene amplification reaction with both primers, 5'EcoPBR and 3'EcoPBR. Prior to amplification, the components are incubated at 95 ° C for 4 minutes. The gene amplification reaction is performed at 94 ° C. for 1 minute, 68 ° C. for 1 minute and 72 ° C. for 2 minutes for 30 cycles, and then extended at 72 ° C. for 5 minutes.
[0078]
Genomic DNA is available from Turano, F .; J. et al. (1992) Plant Physilo. Isolate from Arabidopsis thaliana leaves at day 24 as described at 100: 374. In the PCR reaction, 250 ng of each primer (5 'EcoPBR and 3' EcoPBR) and 500 ng of genomic DNA are used. The components are incubated at 95 ° C. for 10 minutes before the amplification reaction. The gene amplification reaction is performed at 94 ° C for 1 minute, 70 ° C for 1 minute and 72 ° C for 3 minutes for 30 cycles, and then extended at 72 ° C for 5 minutes.
[0079]
Genomic DNA and cDNA fragments are separately cloned into PCR2.1 (Invitrogen Corp. Carlsbad, CA, USA) and sequenced by the Taq Diodexy terminator cycle sequence (Applied Biosystems) method. Data is analyzed on a Power Macintosh 6500/250 with MacVector software.
Embodiment 3
[0080]
Construction of transgenic plants
Transgenic plants that overexpress the plant receptor protein or those that overexpress the antisense receptor protein are prepared as follows. Full (591 bp) open reading of the sense (overexpression) or antisense (insufficient expression) of the receptor protein using conventional cloning techniques to generate PCR, RT-PCR or antisense constructs The frame, or a portion thereof about 25 base pairs in size (for antisense or RNAi only), is cloned into a vector such as pBI121 (Clonetech, Palo Alto, CA). Synthesized gene-specific primers,
[0081]
Embedded image

[0082]
and
[0083]
Embedded image

[0084]
(GenBank, unknown protein, gene # At2g47770, protein id = AAC63632.1, db xref = GI: corresponds to 3738290) is commercially available (Biosynthesis, Inc. Lewisville, Tex.) and is used in RT-PCR reactions. For example, a PCR reaction uses 250 ng of each primer and about 500 ng of genomic DNA. The components are incubated at 95 ° C. for 2 minutes before the amplification reaction. The gene amplification reaction is performed at 94 ° C for 1 minute, 65 ° C for 1 minute and 72 ° C for 2 minutes for 30 cycles, and then extended at 72 ° C for 4 minutes.
[0085]
RT-PCR uses the 5 'RACE system (Life Technologies, Rockville, MD) or the simpler reverse transcriptase (RT) to identify full-length cDNA clones. Poly (A) isolated from day 2 plants using primer 3'EcoPBR according to the manufacturer's instructions⁺1) First strand cDNA from 1 μg of RNA is synthesized. One fifth of the first strand cDNA synthesis is used as a template in a gene amplification reaction with both primers, 5'EcoPBR and 3'EcoPBR. The components are incubated at 95 ° C. for 2 minutes before amplification. The gene amplification reaction is performed at 94 ° C. for 1 minute, 58 ° C. for 1 minute and 72 ° C. for 2 minutes for 30 cycles, and then extended at 72 ° C. for 5 minutes.
[0086]
Genomic DNA or cDNA fragments are cloned into the plant transformation vector in a sense (forward) or antisense (reverse) orientation depending on the desired result. Vectors can include constitutive promoters, such as the CaMV 35S promoter and nopaline synthase terminator, or other promoters described herein and known in the art. The vector can be modified biologically [Sohal et al. , (1999) Plant Mol. Biol. 41: 75-87] or abiotic stress [Ngai et al. (1997) Plant J. et al. 12: 1021-1034; van Der Krol et al. , (1999) Plant Physiol. 121: 1153-1162; Kucho et al. (1999) Plant Physiol 121: 1329-1338], and / or hormones or other signaling molecules [Chen and Singh (1999) Plant J. et al. 19: 667-677; Lu et al. (1998) J. Am. Biol. Chem. 273: 10120-10131]; Leubner-Metzger et al. (1998) Plant Mol. Biol. 38: 785-795]. The orientation of the cloned construct is confirmed by restriction endonuclease and PCR analysis.
[0087]
Upon completion of cloning, the binary vector was partially modified (ie, the addition of 0.02% (v / v) Silwet to the infiltrate) under reduced pressure infiltration [Bechtold, N .; and Bouchez, D .; (1995) In plant Agrobacterium-mediated transformation of adult Arabidopsis thaliana plants by vacuum infiltration. In Gene transfer to Plants. I. Potrykus and G. Edited by Spanenberg. Springer-Verlag, Heidelberg, pp 19-23] was used to introduce into Agrobacterium- tumefaciens detoxifying strains, such as EHA105, followed by Arabidopsis thaliana (Ws ecotype). Seeds collected from the transformed plants are germinated and selected for kanamycin resistance.
[0088]
While the invention has been described and described in detail in the drawings and foregoing description, the same should be considered as describing features and not as limiting, only the preferred embodiments are illustrated, It is to be understood that what has been described, and that all changes and modifications within the spirit of the invention, are desired to be protected. Further, all references cited herein represent levels in the art and are hereby incorporated by reference in their entirety.
[0089]
While specific aspects of the invention will be pointed out with particularity in the claims, the invention itself, and the manner in which it is made and used, will be described below with reference to the accompanying drawings, which form a part of the present invention. Can be better understood with reference to the description of
[Brief description of the drawings]
[0090]
FIG. 1 shows the proposed role of GABA in plant stress response (hypothetical pathway in which GABA functions as a cell barometer and transducer of environmental stress signals).
FIG. 2 is a graph showing the effect of cyclosporin A on GABA-mediated growth promotion in duckweed, described in more detail in Example 1.
FIG. 3 is a graph showing the effect of spermine on GABA-mediated growth promotion in duckweed and is described in more detail in Example 1.
FIG. 4 is a graph showing the effect of quinine on GABA-mediated growth promotion in duckweed, described in more detail in Example 1.
FIG. 5 is a graph showing the effect of diazepam and PK11195 (isoquinolinecarboxamide) on GABA-mediated growth promotion in duckweed, and is described in more detail in Example 1.

Claims (35)

Introducing a nucleic acid molecule having a nucleotide sequence encoding a plant protein having an amino acid sequence having at least about 70% identity to the amino acid sequence set forth in SEQ ID NO: 2 into a plant cell, wherein the nucleic acid molecule is A method for transforming a plant having a foreign promoter operably linked to the 5 'end of a nucleotide sequence. 2. The method of claim 1, wherein said nucleotide sequence has at least about 80% identity to the nucleotide sequence set forth in SEQ ID NO: 1. 3. The method of claim 2, wherein said nucleotide sequence has at least about 90% identity to the nucleotide sequence set forth in SEQ ID NO: 1. 2. The method of claim 1, wherein said promoter is selected from the group consisting of a constitutive promoter, an inducible promoter and a cell-specific promoter. A method for identifying a plant protein comprising hybridizing a nucleic acid probe having a nucleic acid sequence having at least about 60% identity to the nucleotide sequence set forth in SEQ ID NO: 1 to a plant nucleic acid. 6. The method of claim 5, wherein said probe is about 25 to about 500 nucleotides in length. A method for treating a plant, comprising the following steps:
(A) providing to a plant an introduced nucleic acid molecule having a nucleotide sequence encoding a protein having an amino acid sequence having at least 70% identity to the amino acid sequence set forth in SEQ ID NO: 2; and A) treating the plant with an effective amount of GABA; 8. The method of claim 7, wherein said method comprises expressing said nucleic acid sequence prior to said treating. 8. The method of claim 7, wherein said nucleotide sequence comprises a nucleotide sequence having at least about 80% identity to the nucleotide sequence set forth in SEQ ID NO: 1. 10. The method of claim 9, wherein said nucleotide sequence has at least about 90% identity to the nucleotide sequence set forth in SEQ ID NO: 1. 8. The method of claim 7, wherein said plant is treated with a composition comprising GABA and a GABA agonist. Wherein said agonist is from the group consisting of baclofen, cis-4-aminopent-2-enoic acid, imidazole-4-acetic acid and 4,5,6,7-tetrahydroisoxazole [5,4-c] pyridin-3-ol 12. The method of claim 11, which is selected. The method of claim 7, wherein said introduced nucleic acid molecule further comprises a heterologous promoter operably linked to the 5 'end of said nucleotide sequence. A method for regulating plant metabolism comprising the following steps:
(A) comprising a nucleotide sequence complementary to a nucleotide sequence having at least about 70% identity to the nucleotide sequence set forth in SEQ ID NO: 1, or a nucleotide sequence complementary to an RNA sequence transcribed from said sequence Introducing an antisense nucleic acid molecule into the plant cell (b) culturing the plant cell under conditions effective for hybridization of the antisense nucleotide sequence to the plant nucleic acid. 15. The method of claim 14, wherein each of said nucleotide sequences is about 30 to about 100 nucleotides in length. 15. The method of claim 14, wherein each of said nucleotide sequences is about 30 to about 400 nucleotides in length. A method for expressing a plant protein, comprising the following steps:
(A) introducing into a plant cell an isolated nucleic acid molecule having a nucleotide sequence encoding a plant protein having an amino acid sequence having at least about 70% identity to the amino acid sequence set forth in SEQ ID NO: 2; (B) a step of culturing under conditions for expressing the protein. 18. The method of claim 17, wherein said nucleic acid molecule has a nucleotide sequence having at least about 80% identity to the nucleotide sequence set forth in SEQ ID NO: 1. 18. The method of claim 17, further comprising inserting said nucleotide sequence into a vector prior to said introducing step. 20. The method of claim 19, wherein said vector is a plasmid vector. Plant receptor activity, comprising introducing into a plant cell a nucleic acid molecule having a nucleotide sequence encoding a plant protein having an amino acid sequence having at least about 70% identity to the amino acid sequence set forth in SEQ ID NO: 2 How to modify. An isolated nucleic acid molecule comprising a nucleic acid molecule consisting essentially of a nucleotide sequence encoding a protein, wherein said nucleotide sequence has at least about 70% identity to the amino acid sequence set forth in SEQ ID NO: 2. A nucleic acid molecule encoding a plant protein having a foreign promoter operably linked to the 5 'end of the nucleotide sequence. 23. The molecule of claim 22, wherein said nucleotide sequence consists essentially of a protein-encoding nucleotide sequence having at least about 70% identity to the nucleotide sequence set forth in SEQ ID NO: 1. 24. The molecule of claim 23, wherein said nucleotide sequence consists essentially of a protein-encoding nucleotide sequence having at least about 80% identity to the nucleotide sequence set forth in SEQ ID NO: 1. 23. The molecule of claim 22, wherein said protein comprises an amino acid sequence having at least about 80% identity to the amino acid sequence set forth in SEQ ID NO: 1. A recombinant nucleic acid molecule that includes:
(A) a nucleotide sequence consisting essentially of a nucleotide sequence encoding a protein, which encodes a plant protein having an amino acid sequence having at least about 70% identity to the amino acid sequence set forth in SEQ ID NO: 2 And (b) an exogenous promoter operably linked to the 5 'end of the nucleotide sequence. 27. The molecule of claim 26, wherein said nucleotide sequence is a cDNA sequence. 27. The molecule of claim 26, wherein said protein comprises an amino acid sequence having at least about 90% identity to the amino acid sequence set forth in SEQ ID NO: 2. 27. The molecule of claim 26, wherein said promoter is selected from the group consisting of a constitutive promoter, an inducible promoter and a cell-specific promoter. Plant cells, including:
(A) an introduced nucleic acid molecule having a nucleotide sequence encoding a plant protein having an amino acid sequence having at least about 70% identity to the amino acid sequence set forth in SEQ ID NO: 2; and (b) the nucleotide A foreign promoter operably linked to the 5 'end of the sequence. 31. The plant cell of claim 30, wherein said protein comprises an amino acid sequence having at least about 90% identity to the amino acid sequence set forth in SEQ ID NO: 2. 32. The plant cell of claim 31, wherein said protein comprises the amino acid sequence set forth in SEQ ID NO: 2. Transgenic plants, including:
(A) an introduced nucleic acid molecule encoding a plant protein having an amino acid sequence having at least about 70% identity to the amino acid sequence set forth in SEQ ID NO: 2; and (b) 5 'of the nucleotide sequence A foreign promoter operably linked to a terminus. 34. The transgenic plant of claim 33, wherein said protein comprises an amino acid sequence having at least about 90% identity to the amino acid sequence set forth in SEQ ID NO: 2. 34. The transgenic plant of claim 33, wherein said protein comprises the amino acid sequence set forth in SEQ ID NO: 2.

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