この研究は、オランダ技術財団 STW (12385) オランダ組織科学的研究 (NWO) の一部であるし、するが部分的で、経済情勢の大臣 (MS に OTP グラント 12385) 資金を供給によってサポートされます。 我々 は pCH20、BIBAC GW ベクトルのバックボーンを提供するキャロル ・ マクゴーギー ・ ハミルトン (コーネル大学、米国) をありがちましょう。

<ol>
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著者はない競合する金銭的な利益やその他の利害の関係を宣言します。

生成する重要な遺伝子組み換え transgene の 1 つ、そのまま統合は使用バイナリ ベクトルの選択です。BIBAC 家族のベクトルは、種植物多く23,24,25,26,27,28に興味のシーケンスを提供する使用されています。BIBAC ベクトル、BIBAC-GW を含む高効率単一コ?...

遺伝子組換え植物を生成、するとき通常目的は安定を表明した統合された transgene(s) を持つことです。これは、そのままシングル コピー transgene の統合によって達成できます。複数の統合は、遺伝子サイレンシングにも、遺伝子の発現増加につながることができます。遺伝子のサイレンシングは挿入されたシーケンスはタンデムまたは反転繰り返し1,2,3,4に配置された場合に生じやすい。バイナリのベクトル、アグロバクテリウムのシャトルとして使用されます-植物ゲノムに興味のシーケンスを提供する変換実験を介する。植物のゲノムに統合の数はアグロバクテリウム5,6.にバイナリのベクトルのコピー数に依存多くの一般的に使用されるバイナリ ベクトル高いコピー ベクトル、したがって高の平均遺伝子コピー数をもたらす:シロイヌナズナ53.3 4.9 にコピーします。
T DNA の統合の数は、BIBAC7などまたはA. 根頭がんしゅ病菌染色体5T DNA を起動することにより、 A. 根頭がんしゅ病菌で低コピー数を持つバイナリのベクトルを使用して下げることが。このような場合の transgene の統合の平均数は 25,8,9,10以下です。あることが原因シングル コピー A. 根頭がんしゅ病菌で、大腸菌、BIBAC 誘導体が維持し、150 kb11と同じ大きさの構造を提供します。
GW 互換 BIBAC ベクトル10、12は、ゲートウェイのクローン作成を使用してベクターに興味の遺伝子の簡単な紹介を許可します。ゲートウェイ技術を使用するクローンの手順を大幅に簡略化、大規模な低コピー数ベクトル13,14, 低 DNA 収量などユニークな制限の限られた選択に関連付けられている一般的な問題の解決にも7,11のクローンを作成できるサイト。グルホシネート アンモニウム (pBIBAC-バー-GW) にどちらかの抵抗性または種皮 (pBIBAC RFP GW) 植物 (図 1)10,12で選択した DsRed 蛍光 pBIBAC GW 誘導体があります。両方のベクトルのカナマイシン耐性遺伝子は、細菌の選択マーカーとして使用されます。
PBIBAC GW ベクトルを組み合わせる: (1) 簡単なデザインと大腸菌、および (2) そのままシングル コピー統合planta で高効率で遺伝子操作。単一を運ぶ遺伝子組換え植物のおよそ半分とシロイヌナズナにおける平均 1.7 インテグレーションの pBIBAC GW ベクトル収量は T-DNA10を統合されています。
遺伝子を安定に発現は、ほとんど遺伝子組み換え生成のための要件です。遺伝子発現の安定は、そのまま、単一コピーの統合によって実現できます。そのまま、単一コピーの統合を運ぶ遺伝子組換え植物の使用は、しかし、さらに重要な場合たとえば、目的これらの依存性や修復、組み換え、変異などのクロマチンによるプロセスの効率化を研究することですゲノムの位置と挿入部位のクロマチン構造上のプロセス。我々 の関心ローカル ゲノム コンテキストに関する監督オリゴヌクレオチド変異 (ODM) の依存性を研究する突然変異遺伝子のままに、単一コピーの統合記者行のセットだった生成された (図 2)10。この行セットを使用して、むしろ似ている遺伝子発現レベルにもかかわらず、ゲノムの異なる場所で統合された形質転換における遺伝子座間の ODM 効率が異なることが示されました。

<table><tbody><tr><td>Kanamycin sulphate monohydrate</td><td>Duchefa</td><td>K0126</td><td></td></tr><tr><td>Gentamycin sulphate</td><td>Duchefa</td><td>G0124</td><td></td></tr><tr><td>Rifampicin</td><td>Duchefa</td><td>R0146</td><td></td></tr><tr><td>Tetracycline hydrochloride</td><td>Sigma</td><td>T-3383</td><td></td></tr><tr><td>DB3.1 competent cells</td><td>Thermo Scientific - Invitrogen</td><td>11782-018</td><td>One Shot &lt;em&gt;ccdB&lt;/em&gt; Survival 2 T1R Competent Cells (A10460) by Invitrogen or any other ccdB resistant &lt;em&gt;E. coli&lt;/em&gt; strain can be used instead&amp;nbsp;&amp;nbsp;</td></tr><tr><td>DH10B competent cells</td><td>Thermo Scientific - Invitrogen</td><td>18290-015</td><td></td></tr><tr><td>Gateway LR clonase enzyme mix&amp;nbsp;</td><td>Thermo Scientific - Invitrogen</td><td>11791-019</td><td></td></tr><tr><td>tri-Sodium citrate dihydrate</td><td>Merck</td><td>106432</td><td></td></tr><tr><td>Trizma base</td><td>Sigma-Aldrich</td><td>T1503</td><td></td></tr><tr><td>EDTA disodium dihydrate</td><td>Duchefa</td><td>E0511</td><td></td></tr><tr><td>Proteinase K</td><td>Thermo Scientific&amp;nbsp;</td><td>EO0491</td><td></td></tr><tr><td>Bacto tryptone</td><td>BD</td><td>211705</td><td></td></tr><tr><td>Yeast extract</td><td>BD</td><td>212750</td><td></td></tr><tr><td>Sodium chloride</td><td>Honeywell Fluka</td><td>13423</td><td></td></tr><tr><td>Potassium chloride</td><td>Merck</td><td>104936</td><td></td></tr><tr><td>D(+)-Glucose monohydrate</td><td>Merck</td><td>108346</td><td></td></tr><tr><td>Electroporation Cuvettes, 0.1 cm gap</td><td>Biorad</td><td>1652089</td><td></td></tr><tr><td>Electroporator Gene Pulser</td><td>BioRad</td><td></td><td></td></tr><tr><td>Magnesium sulfate heptahydrate</td><td>Calbiochem</td><td>442613</td><td></td></tr><tr><td>D(+)-Maltose monohydrate 90%</td><td>Acros Organics</td><td>32991</td><td></td></tr><tr><td>Sucrose</td><td>Sigma-Aldrich</td><td>84100</td><td></td></tr><tr><td>Silwet L-77</td><td>Fisher Scientific</td><td>NC0138454</td><td></td></tr><tr><td>Murashige Skoog medium</td><td>Duchefa</td><td>M0221</td><td></td></tr><tr><td>Agar</td><td>BD</td><td>214010</td><td></td></tr><tr><td>Glufosinate-ammonium (Basta)</td><td>Bayer</td><td>79391781</td><td></td></tr><tr><td>Restriction enzymes</td><td>NEB</td><td></td><td></td></tr><tr><td>Ethidium Bromide</td><td>Bio-Rad</td><td>1610433</td><td></td></tr><tr><td>Electrophoresis system</td><td>Bio-Rad</td><td></td><td></td></tr><tr><td>Sodium hydroxide</td><td>Merck</td><td>106498</td><td></td></tr><tr><td>Hydrochloric acid</td><td>Merck</td><td>100316</td><td></td></tr><tr><td>Blotting nylon membrane Hybond N+</td><td>Sigma Aldrich</td><td>15358</td><td>or GE Healthcare Life Sciences (RPN203B)</td></tr><tr><td>Whatman 3MM Chr blotting paper</td><td>GE Healthcare Life Sciences</td><td>3030-931</td><td></td></tr><tr><td>dNTP</td><td>Thermo Fisher</td><td>R0181</td><td></td></tr><tr><td>Acetylated BSA</td><td>Sigma-Aldrich</td><td>B2518</td><td></td></tr><tr><td>HEPES</td><td>Sigma-Aldrich</td><td>H4034</td><td></td></tr><tr><td>2-Mercaptoethanol</td><td>Merck</td><td>805740</td><td></td></tr><tr><td>Sephadex G-50 Coarse</td><td>GE Healthcare Life Sciences</td><td>17004401</td><td>or Sephadex G-50 Medium (17004301)</td></tr><tr><td>Dextran sulfate sodium salt</td><td>Sigma-Aldrich</td><td>D8906</td><td></td></tr><tr><td>Sodium Dodecyl Sulfate&amp;nbsp;</td><td>US Biological</td><td>S5010</td><td></td></tr><tr><td>Salmon Sperm DNA</td><td>Sigma-Aldrich</td><td>D7656</td><td></td></tr><tr><td>Sodium dihydrogen phosphate monohydrate</td><td>Merck</td><td>106346</td><td></td></tr><tr><td>Storage Phosphor screen and casette</td><td>GE Healthcare Life Sciences</td><td>28-9564-74</td><td></td></tr><tr><td>Phosphor imager</td><td>GE Healthcare Life Sciences</td><td>Typhoon FLA 7000</td><td></td></tr><tr><td>UV Crosslinker</td><td>Stratagene</td><td>Stratalinker 1800</td><td></td></tr><tr><td>cling film (Saran wrap)</td><td>Omnilabo</td><td>1090681</td><td></td></tr><tr><td>Agarose</td><td>Thermo Scientific - Invitrogen</td><td>16500</td><td></td></tr><tr><td>Boric acid</td><td>Merck</td><td>100165</td><td></td></tr><tr><td>DNA marker &amp;lsquo;Blauw&amp;rsquo;; DNA ladder.</td><td>MRC Holland</td><td>MCT8070</td><td></td></tr><tr><td>DNA marker &amp;lsquo;Rood&amp;rsquo;; DNA ladder</td><td>MRC Holland</td><td>MCT8080</td><td></td></tr><tr><td>Hexanucleotide Mix</td><td>Roche</td><td>11277081001</td><td></td></tr><tr><td>Large-Construct Kit</td><td>Qiagen</td><td>12462</td><td></td></tr><tr><td>Heat-sealable polyethylene tubing, clear</td><td>various providers</td><td></td><td>the width of the tubing should be wider than that of blotting membrane</td></tr><tr><td>Heat sealer</td><td></td><td></td><td></td></tr><tr><td>Membrane filter disk</td><td>Merck</td><td>VSWP02500</td><td></td></tr><tr><td>Magnesium chloride</td><td>Merck</td><td>105833</td><td></td></tr><tr><td>Hybridization mesh</td><td>GE Healthcare Life Sciences</td><td>RPN2519</td><td></td></tr></tbody></table>

generating transgenic plants with single-copy insertions using bibac-gw binary vector

遺伝子組換え植物を生成、するとき一般に目的は transgene を安定に発現をしています。複数コピーの統合遺伝子サイレンシングにさらされている多くの場合と、遺伝子の 1 つ、そのまま統合が必要です。他の pBIBAC の誘導体のような細菌人工染色体 (pBIBAC GW)、に基づいてゲートウェイ互換性がある二進ベクトルは、高効率で単一コピーの遺伝子の挿入をことができます。、元の pBIBAC に改善は興味のシーケンス組み込むことが今簡単にベクトル転送 DNA (T-DNA) ゲートウェイを複製して、ゲートウェイ カセットは pBIBAC GW にクローン化されています。一般的 pBIBAC GW と変換結果 0.2 – 0.5 の効率 %、T DNA のままのシングル コピー統合を運ぶ、遺伝子組み換えの半分という。グルホシネート アンモニウム抵抗または種皮の植物では、選択した DsRed 蛍光と細菌の選択項目としてカナマイシン抵抗 pBIBAC GW ベクトルがあります。ここでは、一連のプロトコルが表示 pBIBAC GW を用いたトランスジェニック植物を生成するプロセスを介してリーダーを指導: 選択するの変換を植物の pBIBAC GW ベクトルに興味のシーケンスを再結合から始まってアグロバクテリウム、各種、遺伝子組み換え、DNA にしみが付くことを使用して挿入のイヌイットとコピーの数のための植物をテストします。注目は、単一および複数の座位でシングルとマルチ コピー統合を認識する DNA あぶらとり戦略の設計に与えられます。

生成された10BIBAC GW システムを使用して、植物の ODM を勉強するため記者構造だった構造は、ゲートウェイ エントリ ベクトル力がある pENTR gm12に設計され、pBIBAC-バー-GW (図 1) ゲートウェイ LR 再結合反応を利用した挿入します。
シロイヌナズナの pDM19、位置 120 eYFP ...

Watch this Scientific Journal Video about Generating Transgenic Plants with Single-copy Insertions Using BIBAC-GW Binary Vector at JoVE.com

Generating Transgenic Plants with Single-copy Insertions Using BIBAC-GW Binary Vector

PBIBAC GW バイナリ ベクトルを使用そのままシングル コピー挿入、簡単なプロセスを生成する遺伝子組換え植物になります。ここでは、一連のプロトコルは、形質転換シロイヌナズナ植物を生成し、イヌイットのための植物をテストのプロセスを介してリーダーを指導し、挿入の数がコピーされます。

BIBAC GW バイナリ ベクトルを使用してシングル コピー挿入と遺伝子組換え植物を生成します。

When generating transgenic plants, generally the objective is to have stable expression of a transgene. This requires a single, intact integration of the ...

generating-transgenic-plants-with-single-copy-insertions-using-bibac

Research

JoVE Journal

Genetics

12.4K ビュー.  University of Amsterdam. PBIBAC GW バイナリ ベクトルを使用そのままシングル コピー挿入、簡単なプロセスを生成する遺伝子組換え植物になります。ここでは、一連のプロトコルは、形質転換シロイヌナズナ植物を生成し、イヌイットのための植物をテストのプロセスを介してリーダーを指導し、挿入の数がコピーされます。

ビデオ: Generating Transgenic Plants with Single-copy Insertions Using BIBAC-GW Binary Vector

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

Modifying Baculovirus Expression Vectors to Produce Secreted Plant Proteins in Insect Cells

It has been a challenge for scientists to express recombinant secretory eukaryotic proteins for structural and biochemical studies. The baculovirus-mediated insect cell expression system is one of the systems used to express recombinant eukaryotic secretory proteins with some post-translational modifications. The secretory proteins need to be routed through the secretory pathways for protein glycosylation, disulfide bonds formation, and other post-translational modifications. To improve the existing insect cell expression of secretory plant proteins, a baculovirus expression vector is modified by the addition of either a GP67 or a hemolin signal peptide sequence between the promoter and multiple-cloning sites. This newly designed modified vector system successfully produced a high yield of soluble recombinant secreted plant receptor proteins of Arabidopsis thaliana. Two of the expressed plant proteins, the extracellular domains of Arabidopsis TDR and PRK3 plasma membrane receptors, were crystallized for X-ray crystallographic studies. The modified vector system is an improved tool that can potentially be used for the expression of recombinant secretory proteins in the animal kingdom as well.

Here, we present the protocols for utilizing the insect cell and baculovirus protein expression system to produce large quantities of plant secreted proteins for protein crystallization. A baculovirus expression vector has been modified with either GP67 or insect hemolin signal peptide for plant protein secretion expression in insect cells.

植物昆虫細胞でのタンパク質を分泌生産するバキュロ ウイルス発現ベクターを変更します。

It has been a challenge for scientists to express recombinant secretory eukaryotic proteins for structural and biochemical studies. The ...

生化学

Inducible, Cell Type-Specific Expression in Arabidopsis thaliana Through LhGR-Mediated Trans-Activation

Inducible, tissue-specific expression is an important and powerful tool to study the spatio-temporal dynamics of genetic perturbation. Combining the flexible and efficient GreenGate cloning system with the proven and benchmarked LhGR system (here termed GR-LhG4) for the inducible expression, we have generated a set of transgenic Arabidopsis lines that can drive the expression of an effector cassette in a range of specific cell types in the three main plant meristems. To this end, we chose the previously developed GR-LhG4 system based on a chimeric transcription factor and a cognate pOp-type promoter ensuring tight control over a wide range of expression levels. In addition, to visualize the expression domain where the synthetic transcription factor is active, an ER-localized mTurquoise2 fluorescent reporter under control of the pOp4 or pOp6 promoter is encoded in driver lines. Here, we describe the steps necessary to generate a driver or effector line and demonstrate how cell type specific expression can be induced and followed in the shoot apical meristem, the root apical meristem and the cambium of Arabidopsis. By using several or all driver lines, the context specific effect of expressing one or multiple factors (effectors) under control of the synthetic pOp promoter can be assessed rapidly, for example in F1 plants of a cross between one effector and multiple driver lines. This approach is exemplified by the ectopic expression of VND7, a NAC transcription factor capable of inducing ectopic secondary cell wall deposition in a cell autonomous manner.

Inducible, Cell Type-Specific Expression in Arabidopsis thaliana Through LhGR-Mediated Trans-Activation

Here, we describe the generation and application of a set of transgenic Arabidopsis thaliana lines enabling inducible, tissue-specific expression in the three main meristems, the shoot apical meristem, the root apical meristem, and the cambium.

誘導、細胞型における発現シロイヌナズナLhGR を介したトランス-活性化

Inducible, tissue-specific expression is an important and powerful tool to study the spatio-temporal dynamics of genetic perturbation. Combining the ...

遺伝学

Rapid Assembly of Multi-Gene Constructs using Modular Golden Gate Cloning

The Golden Gate cloning method enables the rapid assembly of multiple genes in any user-defined arrangement. It utilizes type IIS restriction enzymes that cut outside of their recognition sites and create a short overhang. This modular cloning (MoClo) system uses a hierarchical workflow in which different DNA parts, such as promoters, coding sequences (CDS), and terminators, are first cloned into an entry vector. Multiple entry vectors then assemble into transcription units. Several transcription units then connect into a multi-gene plasmid. The Golden Gate cloning strategy is of tremendous advantage because it allows scar-less, directional, and modular assembly in a one-pot reaction. The hierarchical workflow typically enables the facile cloning of a large variety of multi-gene constructs with no need for sequencing beyond entry vectors. The use of fluorescent protein dropouts enables easy visual screening. This work provides a detailed, step-by-step protocol for assembling multi-gene plasmids using the yeast modular cloning (MoClo) kit. We show optimal and suboptimal results of multi-gene plasmid assembly and provide a guide for screening for colonies. This cloning strategy is highly applicable for yeast metabolic engineering and other situations in which multi-gene plasmid cloning is required.

The goal of this protocol is to provide a detailed, step-by-step guide for assembling multi-gene constructs using the modular cloning system based on Golden Gate cloning. It also gives recommendations on critical steps to ensure optimal assembly based on our experiences.

モジュラーゴールデンゲートクローニングを用いた多遺伝子構造の迅速な組み立て

The Golden Gate cloning method enables the rapid assembly of multiple genes in any user-defined arrangement. It utilizes type IIS restriction enzymes that ...

生物工学

The MultiBac Protein Complex Production Platform at the EMBL

Proteomics research revealed the impressive complexity of eukaryotic proteomes in unprecedented detail. It is now a commonly accepted notion that proteins in cells mostly exist not as isolated entities but exert their biological activity in association with many other proteins, in humans ten or more, forming assembly lines in the cell for most if not all vital functions.1,2 Knowledge of the function and architecture of these multiprotein assemblies requires their provision in superior quality and sufficient quantity for detailed analysis. The paucity of many protein complexes in cells, in particular in eukaryotes, prohibits their extraction from native sources, and necessitates recombinant production. The baculovirus expression vector system (BEVS) has proven to be particularly useful for producing eukaryotic proteins, the activity of which often relies on post-translational processing that other commonly used expression systems often cannot support.3 BEVS use a recombinant baculovirus into which the gene of interest was inserted to infect insect cell cultures which in turn produce the protein of choice. MultiBac is a BEVS that has been particularly tailored for the production of eukaryotic protein complexes that contain many subunits.4 A vital prerequisite for efficient production of proteins and their complexes are robust protocols for all steps involved in an expression experiment that ideally can be implemented as standard operating procedures (SOPs) and followed also by non-specialist users with comparative ease. The MultiBac platform at the European Molecular Biology Laboratory (EMBL) uses SOPs for all steps involved in a multiprotein complex expression experiment, starting from insertion of the genes into an engineered baculoviral genome optimized for heterologous protein production properties to small-scale analysis of the protein specimens produced.5-8 The platform is installed in an open-access mode at EMBL Grenoble and has supported many scientists from academia and industry to accelerate protein complex research projects.

Protein complexes catalyze key cellular functions. Detailed functional and structural characterization of many essential complexes requires recombinant production. MultiBac is a baculovirus/insect cell system particularly tailored for expressing eukaryotic proteins and their complexes. MultiBac was implemented as an open-access platform, and standard operating procedures developed to maximize its utility.

EMBLでMultiBacタンパク質複合体生産プラットフォーム

Proteomics  research revealed the impressive complexity of eukaryotic proteomes in  unprecedented detail. It is now a commonly accepted notion that ...

生物学

Agroinfiltration and PVX Agroinfection in Potato and Nicotiana benthamiana

Agroinfiltration and PVX agroinfection are two efficient transient expression assays for functional analysis of candidate genes in plants. The most commonly used agent for agroinfiltration is Agrobacterium tumefaciens, a pathogen of many dicot plant species. This implies that agroinfiltration can be applied to many plant species. Here, we present our protocols and expected results when applying these methods to the potato (Solanum tuberosum), its related wild tuber-bearing Solanum species (Solanum section Petota) and the model plant Nicotiana benthamiana. In addition to functional analysis of single genes, such as resistance (R) or avirulence (Avr) genes, the agroinfiltration assay is very suitable for recapitulating the R-AVR interactions associated with specific host pathogen interactions by simply delivering R and Avr transgenes into the same cell. However, some plant genotypes can raise nonspecific defense responses to Agrobacterium, as we observed for example for several potato genotypes. Compared to agroinfiltration, detection of AVR activity with PVX agroinfection is more sensitive, more high-throughput in functional screens and less sensitive to nonspecific defense responses to Agrobacterium. However, nonspecific defense to PVX can occur and there is a risk to miss responses due to virus-induced extreme resistance. Despite such limitations, in our experience, agroinfiltration and PVX agroinfection are both suitable and complementary assays that can be used simultaneously to confirm each other&#39;s results.

Agroinfiltration and PVX Agroinfection in Potato and Nicotiana benthamiana

Agroinfiltration and PVX agroinfection are routine functional assays for transient ectopic expression of genes in plants. These methods are efficient assays in effectoromics strategies (rapid resistance and avirulence gene discovery) and crucial to modern research in molecular plant pathology. They meet the demand for robust high-throughput functional analysis in plants.

ジャガイモとニコティアナベンタミアナにおけるアグロインフィルトレーションとPVXアグロパネクチ

Agroinfiltration and PVX agroinfection are two efficient transient expression assays for functional analysis of candidate genes in plants. The most ...

<meta charset="utf8"></head><body>CRISPRを用いたイネ育種におけるレジスタントスターチの効率的なゲノム編集

Breeding by Design for Functional Rice with Genome Editing Technologies

The conventional approaches to crop breeding, which rely predominantly on time-consuming and labor-intensive methods such as traditional hybridization and mutation breeding, face challenges in efficiently introducing targeted traits and generating diverse plant populations. Conversely, the emergence of genome editing technologies has ushered in a paradigm shift, enabling the precise and expedited manipulation of plant genomes to intentionally introduce desired characteristics. One of the most widespread editing tools is the CRISPR/Cas system, which has been used by researchers to study important biology-related problems. However, the precise and effective workflow of genome editing has not been well-defined in crop breeding. In this study, we demonstrated the entire process of breeding rice varieties enriched with high levels of resistant starch (RS), a functional trait that plays a crucial role in preventing diseases such as diabetes and obesity. The workflow encompassed several key steps, such as the selection of functional SBEIIb gene, designing the single-guide RNA (sgRNA), selecting an appropriate genome editing vector, determining the vector delivery method, conducting plant tissue culture, genotyping mutation and phenotypic analysis. Additionally, the time frame necessary for each stage of the process has been clearly demonstrated. This protocol not only streamlines the breeding process but also enhances the accuracy and efficiency of trait introduction, thereby accelerating the development of functional rice varieties.

<meta charset="utf8"></head><body>著者スポットライト:最適な表現型および農業形質を得るためのCRISPR/Casによるイネ育種の合理化

The protocol describes breeding resistant starch rice varieties by design using genome editing technologies in a precise, efficient, and technically simple way.

ゲノム編集技術による機能性イネの設計育種

The conventional approaches to crop breeding, which rely predominantly on time-consuming and labor-intensive methods such as traditional hybridization and ...

Floral-Dip Transformation of Flax (Linum usitatissimum) to Generate Transgenic Progenies with a High Transformation Rate

Agrobacterium-mediated plant transformation via floral-dip is a widely used technique in the field of plant transformation and has been reported to be successful for many plant species. However, flax (Linum usitatissimum) transformation by floral-dip has not been reported. The goal of this protocol is to establish that Agrobacterium and the floral-dip method can be used to generate transgenic flax. We show that this technique is simple, inexpensive, efficient, and more importantly, gives a higher transformation rate than the current available methods of flax transformation.
In summary, inflorescences of flax were dipped in a solution of Agrobacterium carrying a binary vector plasmid (T-DNA fragment plus the Linum Insertion Sequence, LIS-1) for 1 - 2 min. The plants were laid flat on their side for 24 hr. Then, plants were maintained under normal growth conditions until the next treatment. The process of dipping was repeated 2 - 3 times, with approximately 10 - 14 day intervals between dipping. The T1 seeds were collected and germinated on soil. After approximately two weeks, treated progenies were tested by direct PCR; 2 - 3 leaves were used per plant plus the appropriate T-DNA primers. Positive transformants were selected and grown to maturity. The transformation rate was unexpectedly high, with 50 - 60% of the seeds from treated plants being positive transformants. This is a higher transformation rate than those reported for Arabidopsis thaliana and other plant species, using floral-dip transformation. It is also the highest, which has been reported so far, for flax transformation using other methods for transformation.

Floral-Dip Transformation of Flax Linum usitatissimum to Generate Transgenic Progenies with a High Transformation Rate

Here, we present a protocol to transform flax using Agrobacterium-mediated plant transformation via floral-dip. This protocol is simple to perform and inexpensive, yet yields a higher transformation rate than the current available methods for flax transformation.

亜麻の花-DIP変換（<em&gt;アマ</em&gt;）高転換率のトランスジェニック子孫を生成するために、

Agrobacterium-mediated plant transformation via floral-dip is a widely used technique in the field of plant transformation and has been reported to be ...

BIBAC GW バイナリベクトルを使用してシングルコピー挿入と遺伝子組換え植物を生成します。

要約

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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

植物昆虫細胞でのタンパク質を分泌生産するバキュロウイルス発現ベクターを変更します。

誘導、細胞型における発現シロイヌナズナLhGR を介したトランス-活性化

モジュラーゴールデンゲートクローニングを用いた多遺伝子構造の迅速な組み立て

EMBLでMultiBacタンパク質複合体生産プラットフォーム

ジャガイモとニコティアナベンタミアナにおけるアグロインフィルトレーションとPVXアグロパネクチ

ゲノム編集技術による機能性イネの設計育種

ゲノム編集技術による機能性イネの設計育種

ゲノム編集技術による機能性イネの設計育種

亜麻の花-DIP変換（

BIBAC GW バイナリ ベクトルを使用してシングル コピー挿入と遺伝子組換え植物を生成します。

要約

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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

植物昆虫細胞でのタンパク質を分泌生産するバキュロ ウイルス発現ベクターを変更します。

誘導、細胞型における発現シロイヌナズナLhGR を介したトランス-活性化

モジュラーゴールデンゲートクローニングを用いた多遺伝子構造の迅速な組み立て

EMBLでMultiBacタンパク質複合体生産プラットフォーム

ジャガイモとニコティアナベンタミアナにおけるアグロインフィルトレーションとPVXアグロパネクチ

ゲノム編集技術による機能性イネの設計育種

ゲノム編集技術による機能性イネの設計育種

ゲノム編集技術による機能性イネの設計育種

亜麻の花-DIP変換（

BIBAC GW バイナリベクトルを使用してシングルコピー挿入と遺伝子組換え植物を生成します。

植物昆虫細胞でのタンパク質を分泌生産するバキュロウイルス発現ベクターを変更します。