この作品は、全米科学財団 (NSF-PGRP IOS-1339388) から植物ゲノム研究プログラムによって支えられました。

1. 植物の成長
<ol><li>大豆の 5-10 ダイズ種子 (ウィリアムズ 82) 長日条件 (16 h 1,500 µmol m-2の-1光) カスタム土壌ミックスの 25 ° c の下で温室で 13 cm ポットに種をまく (、1: 土、パーライト、魚雷の砂の比率 1:1)。</li>
</ol>2. DNA のプラスミッドの準備
<ol><li>滅菌ピペット チップやつまようじを使用すると、単一コロニーを大腸菌興味の遺伝子を含んでいるプラスミッドを運ぶ冷凍グリセロール ストックを選ぶし、20 mL ルリア ベルターニカミラ (LB) 液体培地 50 mL のフラスコに適切な抗生物質で予防接種します。</li>
	<li>シェーカーで一晩 37 ° C でフラスコを孵化させなさい。5 分間室温で 12,000 × g の懸濁液を遠心分離し、上清を廃棄して細菌を収集します。抽出・精製プラスミド プラスミド準備キットの製造元の手順を実行します。</li>
</ol>3. プロトプ ラスト単離
<ol><li>10 日間の古い大豆苗 (図 1) から新しく拡張された単葉の葉をカットします。 注: 適切な発達段階で葉の選択は大豆プロトプ ラスト調製成功への鍵です。初期の発達段階で展開直後の葉だけ使用します。成熟した葉、消化する細胞壁と難しくなってください。</li>
	<li>新鮮なかみそりの刃を含む葉と 0.5-1 に残る mm ストリップ、カットから主脈を削除します。 注: 2 つの単葉の葉 10 mL の酵素液で消化は以上 10 変換のため十分なプロトプ ラストを与えます。</li>
	<li>葉が 10 mL を 15 mL チューブに作りたての酵素液 (表 1) にすぐにそして軽くをストリップ鉗子、転送のペアを使用しています。真空潜入葉ストリップ部屋の温度で 15 分。</li>
	<li>室温で 4-6 h の低照度下で穏やかな攪拌 (40 回転) と酵素液で葉の細片を孵化させなさい。プロトプ ラストがリリースされると、酵素液が黄色緑を回すことを確認します。(X10) 顕微鏡下で酵素/プロトプ ラスト ソリューションを確認してください。 注: リリースのプロトプ ラストは球状形、未消化の細胞が不規則なまたは楕円形です。</li>
	<li>そっとして 50 mL のチューブで酵素/プロトプ ラスト溶液 10 mL を転送し、消化を停止するために室温 W5 溶液 (表 1) 10 mL を追加します。穏やかに数回チューブを反転します。未消化の葉身を削除する 50 mL のチューブの上に置かれたきれいな 75 μ m ナイロン メッシュに酵素/プロトプ ラストの溶液を注ぐ穏やか。</li>
	<li>室温で 1-2 分の 50 mL のチューブで 100 x g でプロトプ ラスト酵素/フロースルー ソリューションを遠心します。プロトプ ラストのペレットを乱すことがなく 10 mL の血清ピペットを使用して上清を慎重に取り外します。</li>
	<li>プロトプ ラストを再懸濁し、プロトプ ラスト (x10) 顕微鏡で血球の数を数えることによって 2 x 105 mL-1 4 ° C に冷やした W5 ソリューションでの濃度に希釈します。30 分間氷の上プロトプ ラストをしてください。</li>
	<li>室温で 1-2 分の 100 x グラムの懸濁液を遠心し、原形質体の餌を乱すことがなく 1 mL ピペットを使用した W5 ソリューションを慎重に取り外します。室温で 2 x 105 mL-1濃度で MMG 解決方法 (表 1) プロトプ ラストを再懸濁します。</li>
</ol>4. プロトプ ラスト
<ol><li>プロトプ ラスト (2 x 104プロトプ ラスト 2 x 105 mL-1) の 100 μ 因数は 1.5 mL 低接着マイクロ遠心チューブ用ノーカット 200 μ L ピペット チップを使用してします。ネガティブ コントロールとして機能する 1 つの因数脇を置きます。プロトプ ラストの残りの因数のそれぞれにプラスミド (10-20 μ g) の 10 μ L を追加します。</li>
	<li>ゆっくり 1.5 mL 遠心チューブの内壁に作りたての止め釘の解決 (表 1) の 110 μ L を追加し、軽く反転ソリューションが均質になるまでチューブを回転させてください。</li>
	<li>15 分間室温で変換の混合物を孵化させなさい。</li>
	<li>変換を停止するには、ゆっくりと室温で 1.5 mL チューブに W5 ソリューションの 400 μ L を追加し、ソリューションが均一になるまでやさしくチューブを転倒させます。室温で 1-2 分の 100 g でチューブを遠心分離、ピペットを使用して上澄みを廃棄します。</li>
	<li>チューブに無線ソリューション (表 1) の 1 つの mL を追加し、1-2 回を軽くピペッティングによって再懸濁します。表面をコートし、プロトプ ラストが板にくっつかないように 6 ウェル培養プレートの各ウェルに 5% (巻/巻) 滅菌子牛血清 1 mL を追加します。</li>
	<li>数秒後、ピペットを使用して子牛血清を破棄します。再懸濁のプロトプ ラスト培養プレートのウェルに移転します。蓋付きプレートをカバーします。</li>
</ol>5. プロトプ ラストの培養と収穫
<ol><li>暗闇の中で 1-2 日間室温でプロトプ ラストを孵化させなさい。 注: 1 日培養に比べて一般に強い蛍光蛋白質信号を 2 日間培養に利回りし、蛍光蛋白質信号が 3-4 日間続く可能性がありますがわかった。</li>
	<li>プロトプ ラストのソリューションを 1.5 mL の低接着微量遠心チューブに転送します。プロトプ ラストを収穫するために室温で 1-2 分の 100 x g でチューブを遠心します。ピペットを使用して上清を除去し、スライド ガラスに 10 μ L プロトプ ラストを転送します。</li>
	<li>蛍光共焦点顕微鏡又は蛍光信号を観察します。(信号を守らなければなりませんない) 陰性対照として非変形プロトプ ラストを使用します。</li>
</ol>

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A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+NAC+domain-containing+protein,+GmNAC6,+is+a+downstream+component+of+the+ER+stress-and+osmotic+stress-induced+NRP-mediated+cell-death+signaling+pathway.">The NAC domain-containing protein, GmNAC6, is a downstream component of the ER stress-and osmotic stress-induced NRP-mediated cell-death signaling pathway.</a> BMC Plant Biol. 11 (1), 129 (2011).</li><li>Kidokoro, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Soybean+DREB1/CBF-type+transcription+factors+function+in+heat+and+drought+as+well+as+cold+stress-responsive+gene+expression.">Soybean DREB1/CBF-type transcription factors function in heat and drought as well as cold stress-responsive gene expression.</a> Plant J. 81 (3), 505-518 (2015).</li><li>Sun, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeted+mutagenesis+in+soybean+using+the+CRISPR-Cas9+system.">Targeted mutagenesis in soybean using the CRISPR-Cas9 system.</a> Sci Rep-UK. 5, 10342 (2015).</li><li>Karimi, M., Inz&#233;, D., Depicker, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GATEWAY&#8482;+vectors+for+Agrobacterium-mediated+plant+transformation.">GATEWAY&#8482; vectors for Agrobacterium-mediated plant transformation.</a> Trends Plant Sci. 7 (5), 193-195 (2002).</li><li>Xia, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Positional+cloning+and+characterization+reveal+the+molecular+basis+for+soybean+maturity+locus+E1+that+regulates+photoperiodic+flowering.">Positional cloning and characterization reveal the molecular basis for soybean maturity locus E1 that regulates photoperiodic flowering.</a> P. Natl. A. Sci. USA. 109 (32), E2155-E2164 (2012).</li></ol>

著者は、彼らは競合する金銭的な利益があることを宣言します。

ダイズプロトプと一過性発現研究への応用の分離のこのプロトコルは徹底的にテストされており、当研究室では非常にうまきます。手順はシンプルで簡単な一般的な機器を最小コストを必要とされます。我々 のプロトコルは、以前に報告された方法34,35,36,37,38に比べて?...

プロトプ ラストは、削除される細胞壁を持つ植物細胞です。機能のほとんどは、植物細胞の活動を管理するのでプロトプ ラスト観察・多様な携帯電話イベントを評価する良いモデル系、1体の交配を研究し、植物の再生2する貴重なツールです。プロトプ ラストは、細胞壁がブロックされセルに DNA の通過以来工場変換3,4、5、また広く利用されています。プロトプ ラストは生理学的な応答のいくつか、したがって細胞内タンパク質局在6,7、8を研究する基礎研究の基本的な値を提供して、そのまま植物の細胞プロセスを所有しています。蛋白質蛋白質の相互作用の9,10、プロモーター活動11,12,13ではセルを住んでいます。
196014で最初に報告された植物プロトプ ラストの分離と分離とプロトプ ラストの変形のためのプロトコルを開発し、最適化します。葉の切断と非消化組織破片からリリースされた原形質体の分離に続いて、細胞壁の酵素消化、原形質体の隔離の標準的な手順が含まれます。エレクトロポレーション15,16、マイクロインジェクション17,18、およびポリエチレング リコール (PEG)4,5,19手法変革戦略が含まれています。種の広い範囲は、原形質体の隔離、柑橘類20、アブラナ科21、ナス科22その他の観賞用植物家族23,24を含む成功報告されています。モデル植物シロイヌナズナの葉から分離したシロイヌナズナ葉肉プロトプ ラスト (TEAMP) に一過性発現系が確立した25をされている様々 な種では、多様な組織型が使用されて、多様なアプリケーションに広く採用されています。
大豆 (最高グリシン(l.)ダイズ) 最も重要なタンパク質の 1 つで、油作物26。異なり、シロイヌナズナイネ、遺伝子組み換え大豆の植物を得ることはむしろ困難と低効率に知られています。アグロバクテリウム-仲介された浸潤一般に使用されているタバコ27で表皮細胞およびシロイヌナズナ28,29, 苗の一時的な遺伝子発現研究に対しサツマイモネコブセンチュウは大豆30植物毛状根の変形のために使用されています。ウイルス誘導性遺伝子のサイレンシング アプローチは、ターゲット遺伝子の31,32と過渡式の33のダウンレギュレーションを系統的に利用されています。プロトプ ラストは、貴重な汎用性の高いこれらのアプローチ方法を提供します。プロトプ ラストは大豆の地上材料から得ることが、発現の迅速かつ同期ができます。しかし、1983年34ダイズプロトプ ラストの最初の成功した分離以来報告されている限られた大豆35,36,37,プロトプ ラストの応用38、主にダイズプロトプ ラストの比較的低い収穫のため。
ここでは、ダイズプロトプ ラストの分離のためのシンプルで効率的なプロトコルと一時的な遺伝子発現研究への応用について述べる。ダイズ芽ばえから若い単葉の葉を使用すると、数時間以内の重要なプロトプ ラストの大量を取得することができました。さらに、ペグによるカルシウムの変換法はシンプルで高効率で大豆プロトプ ラストに DNA を提供する低コストを最適化しています。

<table><tbody><tr><td>MES</td><td>Sigma Aldrich</td><td>M8250-100G</td><td></td></tr><tr><td>Cellulase CELF</td><td>Worthington Biological Corporation</td><td>LS002611</td><td></td></tr><tr><td>Pectolyase Y-23</td><td>BioWorld</td><td>9033-35-6</td><td></td></tr><tr><td>CELLULASE &quot;ONOZUKA&quot; R-10</td><td>yakult</td><td>10g</td><td></td></tr><tr><td>MACEROZYME R-10</td><td>yakult</td><td>10g</td><td></td></tr><tr><td>Mannitol</td><td>ICN Biomedicals</td><td>152540</td><td></td></tr><tr><td>CaCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Fisher</td><td>C79-500g</td><td></td></tr><tr><td>BSA</td><td>NEB</td><td>R3535S</td><td></td></tr><tr><td>DTT</td><td>Sigma Aldrich</td><td>D5545-5G</td><td></td></tr><tr><td>NaCl</td><td>Sigma Aldrich</td><td>S7653-1kg</td><td></td></tr><tr><td>KCl</td><td>Fisher</td><td>P217-500g</td><td></td></tr><tr><td>MgCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Sigma Aldrich</td><td>M8266-100g</td><td></td></tr><tr><td>PEG4000</td><td>Fluka</td><td>81240</td><td></td></tr><tr><td>nylon mesh</td><td>carolina</td><td>652222N</td><td></td></tr><tr><td>Tissue Culture Plates</td><td>USA Scientific</td><td>CC7682-7506</td><td></td></tr><tr><td>Razor Blades</td><td>Fisher</td><td>12-640</td><td></td></tr><tr><td>hemacytometer</td><td>hausserscientific</td><td>1483</td><td></td></tr><tr><td>QIAprep Spin Miniprep Kit</td><td>Qiagen</td><td>27104</td><td></td></tr><tr><td>EZNA plasmid miniprep kit</td><td>Omega</td><td>D6942-01</td><td></td></tr><tr><td>GeneJET Plasmid Miniprep Kit</td><td>Thermo Scientific</td><td>K0502</td><td></td></tr><tr><td>Centrifuge 5810</td><td>eppendorf</td><td>5811000827</td><td></td></tr><tr><td>Centrifuge 5424</td><td>eppendorf</td><td>22620401</td><td></td></tr><tr><td>Jencons Powerpette Plus Pipet Controller</td><td>Jencons</td><td>14526-202</td><td></td></tr><tr><td>Zeiss 710 Confocal Microscope</td><td>Zeiss</td><td>N/A</td><td></td></tr><tr><td>Nonstick, RNase-free Microfuge Tubes, 1.5 mL</td><td>Ambion</td><td>AM12450</td><td></td></tr><tr><td>15 mL Centrifuge Tubes</td><td>Denville</td><td>C1018-P</td><td></td></tr><tr><td>50 mL Centrifuge Tubes</td><td>Denville</td><td>C1060-P</td><td></td></tr><tr><td>Newborn Calf Serum</td><td>Thermo Scientific</td><td>16010159</td><td></td></tr><tr><td>Soil</td><td>Ingram&#039;s Nursery</td><td></td><td></td></tr><tr><td>perlite</td><td>Vigoro</td><td>100521091</td><td></td></tr><tr><td>Torpedo Sand</td><td>JKS Ventures</td><td></td><td></td></tr><tr><td>LB Broth, Lennox (Powder)</td><td>Fisher</td><td>BP1427-500</td><td></td></tr></tbody></table>

a simple method for isolation of soybean protoplasts and application to transient gene expression analyses

大豆 (最高グリシン(l.)ダイズ) は重要な作物を遺伝学的、生化学的経路の研究マメ科のモデルとなっています。そのため、大豆の効率的な一過的遺伝子発現系を確立することが重要です。ダイズプロトプ ラストの準備のための単純なプロトコルと非定常の機能解析への応用を報告します。ダイズ芽ばえから若い単葉の葉が高品質のプロトプ ラストの大量に起因したと分かった。ペグ カルシウム仲介された変形方法を最適化することにより、大豆含むプロトプ ラストを用いた高い変換効率を達成しました。このシステムは、ライブ ダイズ細胞における複雑な規制とシグナル伝達メカニズムの検討の効率的かつ汎用性の高いモデルを提供し、ヘルプは、マメ科植物の多様な細胞・発達・生理学的なプロセスを理解するかもしれない。

プロトプ ラストの調製 (図 1) を 10 日間の古い大豆のさまざまな器官を調べた (図 2) 顕微鏡下では認められなかった.胚軸, 上胚軸から細胞壁がほとんど消化して、そしていくつかの細胞 (図 2 b, 2 C) 互いに接続されています。子葉 (図 2 D) とルート (図 ...

Watch this Scientific Journal Video about A Simple Method for Isolation of Soybean Protoplasts and Application to Transient Gene Expression Analyses at JoVE.com

A Simple Method for Isolation of Soybean Protoplasts and Application to Transient Gene Expression Analyses

我々 は生きているセルで複雑な規制とシグナル伝達メカニズムを研究するダイズプロトプ ラストの大量の準備のためシンプルで効率的なプロトコルを開発しました。

簡便な分離大豆プロトプ ラストと一過的遺伝子発現解析への応用

Soybean (Glycine max (L.) Merr.) is an important crop species and has become a legume model for the studies of genetic and biochemical pathways. ...

a-simple-method-for-isolation-soybean-protoplasts-application-to

Research

JoVE Journal

Biology

24.9K ビュー.  California State University, Northridge. 我々 は生きているセルで複雑な規制とシグナル伝達メカニズムを研究するダイズプロトプ ラストの大量の準備のためシンプルで効率的なプロトコルを開発しました。

ビデオ: A Simple Method for Isolation of Soybean Protoplasts and Application to Transient Gene Expression Analyses

A Robotic Platform for High-throughput Protoplast Isolation and Transformation

Over the last decade there has been a resurgence in the use of plant protoplasts that range from model species to crop species, for analysis of signal transduction pathways, transcriptional regulatory networks, gene expression, genome-editing, and gene-silencing. Furthermore, significant progress has been made in the regeneration of plants from protoplasts, which has generated even more interest in the use of these systems for plant genomics. In this work, a protocol has been developed for automation of protoplast isolation and transformation from a &#39;Bright Yellow&#39; 2 (BY-2) tobacco suspension culture using a robotic platform. The transformation procedures were validated using an orange fluorescent protein (OFP) reporter gene (pporRFP) under the control of the Cauliflower mosaic virus 35S promoter (35S). OFP expression in protoplasts was confirmed by epifluorescence microscopy. Analyses also included protoplast production efficiency methods using propidium iodide. Finally, low-cost food-grade enzymes were used for the protoplast isolation procedure, circumventing the need for lab-grade enzymes that are cost-prohibitive in high-throughput automated protoplast isolation and analysis. Based on the protocol developed in this work, the complete procedure from protoplast isolation to transformation can be conducted in under 4 hr, without any input from the operator. While the protocol developed in this work was validated with the BY-2 cell culture, the procedures and methods should be translatable to any plant suspension culture/protoplast system, which should enable acceleration of crop genomics research.

A high-throughput, automated, tobacco protoplast production and transformation methodology is described. The robotic system enables massively parallel gene expression and discovery in the model BY-2 system that should be translatable to non-model crops.

ハイスループットプロトプラスト単離と変革のためのロボットプラットフォーム

Over the last decade there has been a resurgence in the use of plant protoplasts that range from model species to crop species, for analysis of signal ...

遺伝学

Transient Gene Expression in Tobacco using Gibson Assembly and the Gene Gun

In order to target a single protein to multiple subcellular organelles, plants typically duplicate the relevant genes, and express each gene separately using complex regulatory strategies including differential promoters and/or signal sequences. Metabolic engineers and synthetic biologists interested in targeting enzymes to a particular organelle are faced with a challenge: For a protein that is to be localized to more than one organelle, the engineer must clone the same gene multiple times. This work presents a solution to this strategy: harnessing alternative splicing of mRNA. This technology takes advantage of established chloroplast and peroxisome targeting sequences and combines them into a single mRNA that is alternatively spliced. Some splice variants are sent to the chloroplast, some to the peroxisome, and some to the cytosol. Here the system is designed for multiple-organelle targeting with alternative splicing. In this work, GFP was expected to be expressed in the chloroplast, cytosol, and peroxisome by a series of rationally designed 5&#8217; mRNA tags. These tags have the potential to reduce the amount of cloning required when heterologous genes need to be expressed in multiple subcellular organelles. The constructs were designed in previous work11, and were cloned using Gibson assembly, a ligation independent cloning method that does not require restriction enzymes. The resultant plasmids were introduced into Nicotiana benthamiana epidermal leaf cells with a modified Gene Gun protocol. Finally, transformed leaves were observed with confocal microscopy.

This work describes a novel method for selectively targeting subcellular organelles in plants, assayed using the BioRad Gene Gun.

ギブソンアセンブリと遺伝子銃を用いてタバコの一過性の遺伝子発現

In order to target a single protein to multiple subcellular organelles, plants typically duplicate the relevant genes, and express each gene separately ...

環境学

Isolation of Protoplasts from Tissues of 14-day-old Seedlings of Arabidopsis thaliana

Protoplasts are plant cells that have had their cell walls enzymatically removed. Isolation of protoplasts from different plant tissues was first reported more than 40 years ago 1 and has since been adapted to study a variety of cellular processes, such as subcellular localization of proteins, isolation of intact organelles and targeted gene-inactivation by double stranded RNA interference (RNAi) 2-5. Most of the protoplast isolation protocols use leaf tissues of mature Arabidopsis (e.g. 35-day-old plants) 2-4. We modified existing protocols by employing 14-day-old Arabidopsis seedlings. In this procedure, one gram of 14-day-old seedlings yielded 5 106-107 protoplasts that remain intact at least 96 hours. The yield of protoplasts from seedlings is comparable with preparations from leaves of mature Arabidopsis, but instead of 35-36 days, isolation of protoplasts is completed in 15 days. This allows decreasing the time and growth chamber space that are required for isolating protoplasts when mature plants are used, and expedites the downstream studies that require intact protoplasts.

Isolation of Protoplasts from Tissues of 14-day-old Seedlings of Arabidopsis thaliana

This video shows a procedure for isolating intact protoplasts from tissues of 14-day-old seedlings of Arabidopsis. Given that the isolated protoplasts remain intact for at least 96h and are isolated from seedlings instead of one-month-old mature plants, this procedure expedites assays requiring intact protoplasts.

の14日齢の苗の組織からのプロトプラストの単離シロイヌナズナ</em

Protoplasts are plant cells that have had their cell walls enzymatically removed. Isolation of protoplasts  from different plant tissues was first ...

生物学

Robotics and Dynamic Image Analysis for Studies of Gene Expression in Plant Tissues

Gene expression in plant tissues is typically studied by destructive extraction of compounds from plant tissues for in vitro analyses. The methods presented here utilize the green fluorescent protein (gfp) gene for continual monitoring of gene expression in the same pieces of tissues, over time. The gfp gene was placed under regulatory control of different promoters and introduced into lima bean cotyledonary tissues via particle bombardment. Cotyledons were then placed on a robotic image collection system, which consisted of a fluorescence dissecting microscope with a digital camera and a 2-dimensional robotics platform custom-designed to allow secure attachment of culture dishes. Images were collected from cotyledonary tissues every hour for 100 hours to generate expression profiles for each promoter. Each collected series of 100 images was first subjected to manual image alignment using ImageReady to make certain that GFP-expressing foci were consistently retained within selected fields of analysis. Specific regions of the series measuring 300 x 400 pixels, were then selected for further analysis to provide GFP Intensity measurements using ImageJ software. Batch images were separated into the red, green and blue channels and GFP-expressing areas were identified using the threshold feature of ImageJ. After subtracting the background fluorescence (subtraction of gray values of non-expressing pixels from every pixel) in the respective red and green channels, GFP intensity was calculated by multiplying the mean grayscale value per pixel by the total number of GFP-expressing pixels in each channel, and then adding those values for both the red and green channels. GFP Intensity values were collected for all 100 time points to yield expression profiles. Variations in GFP expression profiles resulted from differences in factors such as promoter strength, presence of a silencing suppressor, or nature of the promoter. In addition to quantification of GFP intensity, the image series were also used to generate time-lapse animations using ImageReady. Time-lapse animations revealed that the clear majority of cells displayed a relatively rapid increase in GFP expression, followed by a slow decline. Some cells occasionally displayed a sudden loss of fluorescence, which may be associated with rapid cell death. Apparent transport of GFP across the membrane and cell wall to adjacent cells was also observed. Time lapse animations provided additional information that could not otherwise be obtained using GFP Intensity profiles or single time point image collections.

We report a method for introduction, tracking and quantitative analysis of GFP expression in plant cells. This method utilizes a custom-designed robotics system for semi-continuous image collection from large numbers of samples, over time. We also demonstrate the use of ImageJ and ImageReady for analysis of image series.

ロボット工学と植物組織における遺伝子発現の研究​​のための動的画像解析

Gene expression in plant tissues is typically studied by destructive extraction of compounds from plant tissues for in vitro analyses. The methods ...

Investigating Tissue- and Organ-specific Phytochrome Responses using FACS-assisted Cell-type Specific Expression Profiling in Arabidopsis thaliana

Light mediates an array of developmental and adaptive processes throughout the life cycle of a plant. Plants utilize light-absorbing molecules called photoreceptors to sense and adapt to light. The red/far-red light-absorbing phytochrome photoreceptors have been studied extensively. Phytochromes exist as a family of proteins with distinct and overlapping functions in all higher plant systems in which they have been studied1. Phytochrome-mediated light responses, which range from seed germination through flowering and senescence, are often localized to specific plant tissues or organs2. Despite the discovery and elucidation of individual and redundant phytochrome functions through mutational analyses, conclusive reports on distinct sites of photoperception and the molecular mechanisms of localized pools of phytochromes that mediate spatial-specific phytochrome responses are limited. We designed experiments based on the hypotheses that specific sites of phytochrome photoperception regulate tissue- and organ-specific aspects of photomorphogenesis, and that localized phytochrome pools engage distinct subsets of downstream target genes in cell-to-cell signaling. We developed a biochemical approach to selectively reduce functional phytochromes in an organ- or tissue-specific manner within transgenic plants. Our studies are based on a bipartite enhancer-trap approach that results in transactivation of the expression of a gene under control of the Upstream Activation Sequence (UAS) element by the transcriptional activator GAL43. The biliverdin reductase (BVR) gene under the control of the UAS is silently maintained in the absence of GAL4 transactivation in the UAS-BVR parent4. Genetic crosses between a UAS-BVR transgenic line and a GAL4-GFP enhancer trap line result in specific expression of the BVR gene in cells marked by GFP expression4. BVR accumulation in Arabidopsis plants results in phytochrome chromophore deficiency in planta5-7. Thus, transgenic plants that we have produced exhibit GAL4-dependent activation of the BVR gene, resulting in the biochemical inactivation of phytochrome, as well as GAL4-dependent GFP expression. Photobiological and molecular genetic analyses of BVR transgenic lines are yielding insight into tissue- and organ-specific phytochrome-mediated responses that have been associated with corresponding sites of photoperception4, 7, 8. Fluorescence Activated Cell Sorting (FACS) of GFP-positive, enhancer-trap-induced BVR-expressing plant protoplasts coupled with cell-type-specific gene expression profiling through microarray analysis is being used to identify putative downstream target genes involved in mediating spatial-specific phytochrome responses. This research is expanding our understanding of sites of light perception, the mechanisms through which various tissues or organs cooperate in light-regulated plant growth and development, and advancing the molecular dissection of complex phytochrome-mediated cell-to-cell signaling cascades.

Investigating Tissue- and Organ-specific Phytochrome Responses using FACS-assisted Cell-type Specific Expression Profiling in Arabidopsis thaliana

The molecular basis of spatial-specific phytochrome responses is being investigated using transgenic plants that exhibit tissue- and organ-specific phytochrome deficiencies. The isolation of specific cells exhibiting induced phytochrome chromophore depletion by Fluorescence-Activated Cell Sorting followed by microarray analyses is being utilized to identify genes involved in spatial-specific phytochrome responses.

でFACSアシスト細胞型特異的発現プロファイリングを使用して組織と器官特異フィトクロム応答を調査シロイヌナズナ</em

Light mediates an array of developmental and adaptive processes throughout the life cycle of a plant. Plants utilize light-absorbing molecules called ...

Identification of Plasmodesmal Localization Sequences in Proteins In Planta

Plasmodesmata (Pd) are cell-to-cell connections that function as gateways through which small and large molecules are transported between plant cells. Whereas Pd transport of small molecules, such as ions and water, is presumed to occur passively, cell-to-cell transport of biological macromolecules, such proteins, most likely occurs via an active mechanism that involves specific targeting signals on the transported molecule. The scarcity of identified plasmodesmata (Pd) localization signals (PLSs) has severely restricted the understanding of protein-sorting pathways involved in plant cell-to-cell macromolecular transport and communication. From a wealth of plant endogenous and viral proteins known to traffic through Pd, only three PLSs have been reported to date, all of them from endogenous plant proteins. Thus, it is important to develop a reliable and systematic experimental strategy to identify a functional PLS sequence, that is both necessary and sufficient for Pd targeting, directly in the living plant cells. Here, we describe one such strategy using as a paradigm the cell-to-cell movement protein (MP) of the Tobacco mosaic virus (TMV). These experiments, that identified and characterized the first plant viral PLS, can be adapted for discovery of PLS sequences in most Pd-targeted proteins.

Identification of Plasmodesmal Localization Sequences in Proteins In Planta

Plant intercellular connections, the plasmodesmata (Pd), play central roles in plant physiology and plant-virus interactions. Critical to Pd transport are sorting signals that direct proteins to Pd. However, our knowledge about these sequences is still in its infancy. We describe a strategy to identify Pd localization signals in Pd-targeted proteins.

タンパク質Planta Plasmodesmal ローカリゼーション シーケンスの同定

Plasmodesmata (Pd) are cell-to-cell connections that function as gateways through which small and large molecules are transported between plant cells. ...

免疫・感染学

Efficient and Rapid Isolation of Early-stage Embryos from Arabidopsis thaliana Seeds

In flowering plants, the embryo develops within a nourishing tissue - the endosperm - surrounded by the maternal seed integuments (or seed coat). As a consequence, the isolation of plant embryos at early stages (1 cell to globular stage) is technically challenging due to their relative inaccessibility. Efficient manual dissection at early stages is strongly impaired by the small size of young Arabidopsis seeds and the adhesiveness of the embryo to the surrounding tissues. Here, we describe a method that allows the efficient isolation of young Arabidopsis embryos, yielding up to 40 embryos in 1 hr to 4 hr, depending on the downstream application. Embryos are released into isolation buffer by slightly crushing 250-750 seeds with a plastic pestle in an Eppendorf tube. A glass microcapillary attached to either a standard laboratory pipette (via a rubber tube) or a hydraulically controlled microinjector is used to collect embryos from droplets placed on a multi-well slide on an inverted light microscope. The technical skills required are simple and easily transferable, and the basic setup does not require costly equipment. Collected embryos are suitable for a variety of downstream applications such as RT-PCR, RNA sequencing, DNA methylation analyses, fluorescence in situ hybridization (FISH), immunostaining, and reporter gene assays.

Efficient and Rapid Isolation of Early-stage Embryos from Arabidopsis thaliana Seeds

We report an efficient and simple method to isolate embryos at early stages of development from Arabidopsis thaliana seeds. Up to 40 embryos can be isolated in 1 hr to 4 hr, depending on the downstream application. The procedure is suitable for transcriptome, DNA methylation, reporter gene expression, immunostaining and fluorescence in situ hybridization analyses.

から初期段階の胚の効率的かつ迅速な単離<em&gt;シロイヌナズナ</em&gt;種子

In flowering plants, the embryo develops within a nourishing tissue - the endosperm - surrounded by the maternal seed integuments (or seed coat). As a ...

遺伝子組み換えダイズ毛根の高効率生産

Soybean Hairy Root Transformation for the Analysis of Gene Function

Soybean (Glycine max) is a valuable crop in agriculture that has thousands of industrial uses. Soybean roots are the primary site of interaction with soil-borne microbes that form symbiosis to fix nitrogen and pathogens, which makes research involving soybean root genetics of prime importance to improve its agricultural production. The genetic transformation of soybean hairy roots (HRs) is mediated by the Agrobacterium rhizogenes strain NCPPB2659 (K599) and is an efficient tool for studying gene function in soybean roots, taking only 2 months from start to finish. Here, we provide a detailed protocol that outlines the method for overexpressing and silencing a gene of interest in soybean HRs. This methodology includes soybean seed sterilization, infection of cotyledons with K599, and the selection and harvesting of genetically transformed HRs for RNA isolation and, if warranted, metabolite analyses. The throughput of the approach is sufficient to simultaneously study several genes or networks and could determine the optimal engineering strategies prior to committing to long-term stable transformation approaches.

著者スポットライト:遺伝子機能解析のためのダイズ毛根形質転換  

Here, we present a protocol for the high-efficiency production of transgenic soybean hairy roots.

遺伝子機能解析のためのダイズ毛状根形質転換

Soybean (Glycine max) is a valuable crop in agriculture that has thousands of industrial uses. Soybean roots are the primary site of interaction with ...

簡便な分離大豆プロトプラストと一過的遺伝子発現解析への応用

要約

さらに動画を探す

ハイスループットプロトプラスト単離と変革のためのロボットプラットフォーム

ギブソンアセンブリと遺伝子銃を用いてタバコの一過性の遺伝子発現

の14日齢の苗の組織からのプロトプラストの単離シロイヌナズナ

ロボット工学と植物組織における遺伝子発現の研究のための動的画像解析

でFACSアシスト細胞型特異的発現プロファイリングを使用して組織と器官特異フィトクロム応答を調査シロイヌナズナ

タンパク質Planta Plasmodesmal ローカリゼーションシーケンスの同定

から初期段階の胚の効率的かつ迅速な単離

遺伝子機能解析のためのダイズ毛状根形質転換

遺伝子機能解析のためのダイズ毛状根形質転換

遺伝子機能解析のためのダイズ毛状根形質転換

簡便な分離大豆プロトプ ラストと一過的遺伝子発現解析への応用

要約

さらに動画を探す

ハイスループットプロトプラスト単離と変革のためのロボットプラットフォーム

ギブソンアセンブリと遺伝子銃を用いてタバコの一過性の遺伝子発現

の14日齢の苗の組織からのプロトプラストの単離シロイヌナズナ

ロボット工学と植物組織における遺伝子発現の研究​​のための動的画像解析

でFACSアシスト細胞型特異的発現プロファイリングを使用して組織と器官特異フィトクロム応答を調査シロイヌナズナ

タンパク質Planta Plasmodesmal ローカリゼーション シーケンスの同定

から初期段階の胚の効率的かつ迅速な単離

遺伝子機能解析のためのダイズ毛状根形質転換

遺伝子機能解析のためのダイズ毛状根形質転換

遺伝子機能解析のためのダイズ毛状根形質転換

簡便な分離大豆プロトプラストと一過的遺伝子発現解析への応用

ロボット工学と植物組織における遺伝子発現の研究のための動的画像解析

タンパク質Planta Plasmodesmal ローカリゼーションシーケンスの同定