这项研究是由美国国家科学基金会资助IOS-1025642（GBM）的支持。我们感谢玛丽亚·哈里森提供ARqua1应变。

1.指南RNA设计载体的构建<ol><li>确定感兴趣的基因的靶序列。有多种适于此步骤13,14在线CRISPR目标调查程序。 注意:在这里，我们使用GN 20 GG靶基序，但根据所使用的应用或载体系统上的其他设计可能是合适的。 </li><li>设计60聚体gRNA寡聚包括由5侧翼靶基序&#39;和3&#39;20-nt的序列所必需的DNA组件的GN 19部。最后60聚体基序是:5&#39;-TCAAGCGAACCAGTAGGCTT-GN 19 -GTTTTAGAGCTAGAAATAGC-3&#39;。 注:标准，脱盐引物，做工精细。 </li><li>制备用于装配的四个的DNA（ 图1）。见DNA序列的补充文件。 <ol><li>消化1 - p201N 5微克:Cas9 10质粒用限制酶的Spe我在1×缓冲液4在37℃下2小时。柱净化消化ccording制造商的指示。重悬〜15微升10毫摩尔Tris-HCl中，并通过紫外分光光度法量化。 </li><li>执行在25℃的第二与在1×缓冲液3.1限制性酶SWA我消化2小时。检查100 - 200纳克在0.8％琼脂糖凝胶，以确认完全消化。 注:正常消化质粒将在14313 BP有一个乐队。不需要的酶和去磷酸化的热灭活:音符。消化的质粒可被储存在-20℃。 </li><li> PCR扩增使用引SWA I_MtU6F / MtU6R分别ScaffoldF / SPE I_ScaffoldR，从市局gRNA班车10质粒的苜蓿 （百万吨）U6启动子和支架的DNA。使用与PCR条件高保真聚合酶:95℃，3分钟; 31个循环（98℃，20秒，60℃15秒，72℃30秒）;和72℃5分钟。 <ol><li>可视化PCR公关〜3微升等分在1％琼脂糖凝胶oducts确认扩增。该MtU6扩增为377 bp和脚手架扩增为106基点。根据制造商的说明列纯化剩余的PCR产物，在-20℃下用紫外分光光度法和存储量化。 </li></ol></li><li>悬浮gRNA寡聚的整个管在实验室级水100微米。加入1μl100μM寡核苷酸的500微升1×缓冲液2.1。拌匀。如果汇集多个寡核苷酸，加入1微升各100μM寡核苷酸为1倍的缓冲管2.1。稀释寡聚可以储存在-20℃。 </li></ol></li><li>编程热循环仪在50℃下保存，使用加热盖。线性化载体，50毫微克稀释gRNA寡（s）和水的MtU6子，12毫微克（0.2皮摩尔）支架，1微升（0.2皮摩尔）（0.2皮摩尔）的结合100纳克（0.011皮摩尔）至体积5微升。加入5微升2个高保真DNA组装预混，拌匀降速。孵育反应在60分钟50℃的热循环仪。然后置于冰上反应。 注意:反应体积可以增加，以适应低DNA产量。 </li><li>变换2微升反应到感受态大肠杆菌细胞使用在LB标准技术和板补充有50毫克的L -1卡那霉素（菅50）。生长铺板细胞在37℃下过夜。保存在-20℃下剩余的DNA装配反应。 </li><li>使用引StUbi3P218R和ISceIR通过PCR殖民地屏幕。用水稀释1剩余的DNA装配反应的等分试样:5。使用1微升作为殖民地屏幕阳性对照，圆形p201N 1纳克:Cas9质粒作为一个没有插入控件。 <ol><li> PCR扩增的条件; 95℃，3分钟; 31个循环（95℃，30秒，58℃，30秒，72℃30秒）;并在72℃下5分钟。可视化在1％琼脂糖凝胶的PCR产物。确保正确插入725 bp和载体机智有一个乐队豪特的插入（ 例如 ，未切向量）将有一个310-bp条带。 </li></ol></li><li>在37℃下在LB菅生长阳性菌落50液体培养过夜，并根据制造商的说明纯化质粒。桑格序列与StUbi3P218R底漆质粒和对齐色谱图的MtU6启动，目标和支架序列，以确保克隆过程中引入了错误。 </li><li>进行消化〜1微克质粒与生态休旅车和麦粒肿 I.诊断消化可视化在0.8％琼脂糖凝胶。片段（血压）为:4192，2001，1743年，1544 1381 995，831，702，460，423，318，174。 </li><li>使用DNA组装构建载体与多个gRNAs。 <ol><li>为了构建具有两个gRNA盒的载体，PCR扩增用引物SWA I_MtU6F / UNS1_ScaffoldR和第二位置gRNA从1纳克每一个相应的引物UNS1_MtU6F / SPE I_ScaffoldR第一位置gRNA1.8 - 质粒步骤1.1构建。使用的PCR条件在步骤1.3.3。可视化在1％琼脂糖凝胶PCR产物的〜3微升等分试样，以确认扩增。扩增子〜500基点。 </li><li>结合并在200微升混合管的未纯化的PCR产物大约等量的（一般为3 - 4微升每个）。进行组装，加入1微升合并的PCR产物，SWA我的50纳克和SPE I线性p201N:Cas9，实验室级水至2.5微升和2.5微升2×高保真DNA装配主混合物。孵育15分钟50℃的热循环仪。 注意:将DNA组件手册建议一个15分钟温育2 - 3片段。 </li><li>转换1 - 2微升到感受态大肠杆菌细胞和板块在LB菅直人50。生长铺板的细胞在37℃下过夜。保存在-20℃剩余的反应。 </li><li>菌落屏幕通过PCR用引物StUbi3P218R和ISceIR用相同的条件与步骤1.6。正确的Clones将有1200 bp的扩增。在菅直人的LB增长阳性克隆50液体培养过夜，净化质粒。 </li><li>与引StUbi3P218R桑格序列质粒（包括第一位置gRNA）和p201R（包括第二位gRNA）。对齐色谱图至MtU6启动，目标和支架序列。诊断消化用Eco RV和麦粒肿我会导致以下片段（以bp）:4192，2476，1743，1544，1381，995，831，702，460，423，318，174粗体片段含有第二gRNA。 </li></ol></li><li>满足所有质量控制的预期后，转化质粒导入发根农杆菌菌株ARqua1 15。加入1μl质粒制备的50微升电感受态细胞和电穿孔的在1毫米反应杯与设置:2.4千伏，25μF，200Ω。收回〜500微升SOC的细胞，在28℃下摇动2小时。板在LB菅直人50和g排在28℃下2天。使用相同的引物和PCR条件如1.3.3执行菌落屏幕。从阳性克隆使甘油原液。 </li></ol> 2.发根变<ol><li>消毒在20％家用漂白剂番茄种子15分钟，用恒定的混合。在层流罩，除去漂白剂和在无菌实验室级水洗涤3次。 </li><li>在GA-7箱板30种子含1的MS培养基（2.22克L- -1 MS盐+ Gamborg维生素10克L -1蔗糖，3克L -1结冷胶，pH值5.8）。发芽在黑暗中为〜2天，然后移至GA-7框的光。生长〜4中的光多天苗。 </li><li>改造前一天，连胜了A.发根坚实的LB菅直人50文化，在28℃下生长过夜。 </li><li>变换的一天，在层流罩组装无菌材料:12毫升培养试管50毫升锥形管，半MS液相（无绞兰克UM），200μM乙酰丁香酮，滤纸，培养皿，镊子和手术刀，移液器和枪头。加入25微升乙酰丁香酮至50ml MS半，倒〜6毫升到每个培养管。 </li><li>使用弯曲的200微升尖刮A.发根细胞从板块和重悬在6毫升½MS液体。涡流管完全悬浮细胞。从每个向量重悬细胞后，测量在600nm的固定波长为1毫升的细胞的光密度（OD）。确保OD为0.2和0.4之间;稀释或添加更多的细胞是必要的。重复每个结构。 </li><li>约2ml半MS液相添加至培养皿用无菌滤纸。从上沾湿滤纸苗和地方消费税子叶。一旦所有子叶已收集，切成远侧约1cm断子叶，导致在子叶片具有两个切割端。添加外植体A.发根农杆菌的解决方案，混合，孵育20分钟偶尔反相。 注意:每个构建大约12植常规使用，但多达80外植体已经在6ml A的接种发根农杆菌的解决方案。 </li><li>在A.发根农杆菌孵育，加滤纸培养皿，一是每个构建物转化。还设置MS半固体介质，没有抗生素，在层流罩干燥。 </li><li>舀出子叶的A.发根农杆菌的解决方案与干燥滤纸无菌镊子和地点。的Petri盖覆盖，以确保组织不干燥而进行下一个转变。在滤纸上，并转移，背面朝上，印迹子叶到½MS培养基。包裹与手术带和板在黑暗中在室温下共同培养2天。 </li><li>共培养，转子叶，背面侧之后，到½MS培养基补充有300mg的L-1替卡西林/克拉维酸（添300，以防止残油的生长UAL A.发根 ）和菅直人50（对于植物的选择），并包裹与手术胶带。保持在荧光灯下培养在室温下用16小时的光周期。 </li><li>后〜1.5 - 2周根至少有2厘米的长度从子叶使用无菌镊子和手术刀切下并转移到½MS菅50添300的介质。转移10到15根到单个板。标记根尖用标记的位置，与手术胶带包裹，并在培养室维持。 </li><li>一周后，转化的根看到的选择性培养基中生长。嘉实转型根的一个子样本使用首选的DNA提取方法提取DNA。注:与子叶板可以保持2 - 3周的收获，在此之后的根长成一团糟，并难以分离。非收获根组织能无限期地保存。多种测定法可以在分离的DNA样品进行s到确定DNA的突变频率和类型。从几个此类测定结果如下所述。 </li></ol>

<ol>
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作者什么都没有透露。

Since DNA assembly is used to recombine any overlapping DNA sequences, this cloning method can be applied to any CRISPR vector construction. Most CRISPR cloning schemes use either gene synthesis of the gRNA, type IIS restriction enzymes17,18, or overlap-extension PCR19. Each of these techniques has inherent advantages and disadvantages, but they typically require multiple hands-on cloning steps. The primary advantage of the cloning method presented here is that the entire process occurs in a single,...

生成具有针对性的CRISPR DNA修改的能力/ Cas9有功能基因组学研究的巨大潜力。还有CRISPR / Cas9系统的两个组件;的Cas9核酸酶，来自金化脓和指示Cas9到目标DNA的网站，1的约100-nt的导的RNA（gRNA）分子衍生。目标识别由第一赋予〜了gRNA，其允许高通量生产靶向载体2,3的20-nt的。可工程改造大多数生物体，已经已经与CRISPR / Cas9技术4,5。 在植物中，组成型启动子，如CaMV 35S启动子，通常用于驱动Cas9核酸6的表达。的gRNAs使用RNA聚合酶III U6或U3启动子限制gRNA的第一基表示到一个G，对于U6，或A为U3，对有效转录。然而RNA聚合酶II舞会oters，这是自由的这些限制，也已经使用7,8-。 不同gRNAs诱导不同的效率DNA突变，并且因此它可以在整个植物转化的投资或设立广泛的表型屏幕之前先验证CRISPR载体是重要的。 CRISPR的瞬时表达构建体在植物中，通过使用例如农杆菌渗入，通常导致DNA修饰的一个较低的频率相比，稳定的植物6，使得突变困难和表型分析不切实际用这样的方法进行检测。所谓毛状根是由于能够在几周内产生大量的独立的，稳定转化的材料，而不是个月稳定的植物一个方便的替代系统。 CRISPR载体在发根诱导9,10 DNA突变非常有效。 DNA组装方法有效地结扎含，可欣赏的DNA片段apping结束11。的一些DNA的装配方法的主要优点是将单链DNA（ 即寡核苷酸）插入组装产品的能力。因为gRNAs仅〜20-nt的长和新的目标可以用合成的寡核苷酸进行，这些DNA装配方法非常适合于CRISPR克隆。这里所述的协议是基于P201系列CRISPR载体已在大豆10，杨树12被成功地使用，现在番茄。提出的克隆程序在当前克隆方法10提供了几个优点。即，可以在一个单一的一天的单个克隆反应中产生完全功能的载体。载体的构建也可以合并，以产生多个并行CRISPR载体，进一步减少操作时间和材料成本。我们还提出了一个协议番茄毛根的产生作为一种有效的方法，以产生具有目标基因缺失的转基因材料。发根是用来有效吃了CRISPR载体和为后续实验提供材料。

<table><tbody><tr><td>NEBuilder&amp;reg; (HiFi DNA assembly mix)</td><td>New England Biolabs</td><td>E5520</td><td></td></tr><tr><td>p201N:Cas9</td><td>Addgene</td><td>59175</td><td>The p201H:Cas9 plasmid (59176) is also compatible with the reported overlaps and enzymes.</td></tr><tr><td>pUC gRNA Shuttle</td><td>Addgene</td><td>47024</td><td></td></tr><tr><td>&lt;em&gt;Swa&lt;/em&gt;I</td><td>New England Biolabs</td><td>R0604S</td><td></td></tr><tr><td>&lt;em&gt;Spe&lt;/em&gt;I</td><td>New England Biolabs</td><td>R0133S</td><td></td></tr><tr><td>Zymo clean and concentrator-5 column purification</td><td>Zymo Research</td><td>D4003</td><td></td></tr><tr><td>NEB Buffer 2.1</td><td>New England Biolabs</td><td>B7202S</td><td></td></tr><tr><td>NEB CutSmart (Buffer 4)</td><td>New England Biolabs</td><td>B7204S</td><td></td></tr><tr><td>NEB Buffer 3.1</td><td>New England Biolabs</td><td>B7203S</td><td></td></tr><tr><td>EconoSpin Mini Spin Column (plasmid prep)</td><td>Epoch Life Sciences</td><td>1910-050/250</td><td></td></tr><tr><td>EcoRV-HF&amp;reg;</td><td>New England Biolabs</td><td>R3195S</td><td></td></tr><tr><td>StyI-HF&amp;reg;</td><td>New England Biolabs</td><td>R3500S</td><td></td></tr><tr><td>MS Salts + Gamborg Vitamins</td><td>Phytotechnology Laboratories</td><td>M404</td><td></td></tr><tr><td>Phytagel&amp;trade; (gellan gum)</td><td>Sigma Aldrich</td><td>P8169</td><td></td></tr><tr><td>GA-7 Boxes</td><td>Sigma Aldrich</td><td>V8505</td><td></td></tr><tr><td>Micropore&amp;trade; surgical tape</td><td>3M</td><td>1535-0</td><td></td></tr><tr><td>Timentin&amp;reg; (Ticarcillin/Clavulanic acid)</td><td>Various</td><td>0029-6571-26</td><td></td></tr><tr><td>Primers 5&#039; &amp;#8594; 3&#039;</td><td></td><td></td><td></td></tr><tr><td>SwaI_MtU6F&amp;nbsp;</td><td>&lt;strong&gt;GATATTAATCTCTTCGATGA&lt;br/&gt;AATTT&lt;/strong&gt;ATGCCTATCTTATAT&lt;br/&gt;GATCAATGAGG</td><td></td><td></td></tr><tr><td>MtU6R&amp;nbsp;&amp;nbsp;</td><td>AAGCCTACTGGTTCGCTTG&lt;br/&gt;AAG</td><td></td><td></td></tr><tr><td>ScaffoldF&amp;nbsp;</td><td>GTTTTAGAGCTAGAAATAGC&lt;br/&gt;AAGTT</td><td></td><td></td></tr><tr><td>&lt;em&gt;Spe&lt;/em&gt;I_Scaffold R</td><td>&lt;strong&gt;GTCATGAATTGTAATACGACTC&lt;br/&gt;A&lt;/strong&gt;AAAAAAAGCACCGACTCGGTG</td><td></td><td></td></tr><tr><td>StUbi3P218R&amp;nbsp;</td><td>ACATGCACCTAATTTCACTA&lt;br/&gt;GATGT</td><td></td><td></td></tr><tr><td>ISceIR</td><td>GTGATCGATTACCCTGTTAT&lt;br/&gt;CCCTAG</td><td>Cannot be used for Sanger sequencing since there is a second binding site on the plasmid</td><td></td></tr><tr><td>UNS1_Scaffold R&amp;nbsp;</td><td>GAGAATGGATGCGAGTAATGAA&lt;br/&gt;AAAAAGCACCGACTCGGTG</td><td></td><td></td></tr><tr><td>UNS1_MtU6 F&amp;nbsp;&amp;nbsp;</td><td>CATTACTCGCATCCATTCTCAT&lt;br/&gt;GCCTATCTTATATGATCAATGAGG</td><td></td><td></td></tr><tr><td>p201R&amp;nbsp;</td><td>CGCGCCGAATTCTAGTGATCG</td><td></td><td></td></tr><tr><td>Bolded sequences denote 20-nt overlaps with linearized p201N:Cas9.</td><td></td><td></td><td></td></tr></tbody></table>

high-throughput crispr vector construction and characterization of dna modifications by generation of tomato hairy roots

Targeted DNA mutations generated by vectors with clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology have proven useful for functional genomics studies. While most cloning strategies are simple to perform, they generally use multiple steps and can require several days to generate the ultimate constructs. The method presented here is based on DNA assembly and can produce fully functional CRISPR vectors in a single cloning reaction. Vector construction can also be pooled, further increasing the efficiency and utility of the process. A modification of the method is used to create CRISPR vectors with multiple gene targets. CRISPR vectors are then transformed into tomato hairy roots to generate transgenic materials with targeted DNA modifications. Hairy roots are a useful system for testing vector functionality as they are technically simple to generate and amenable to large-scale production. The methods presented here will have wide application as they can be used to generate a variety of CRISPR vectors and be used in a wide range of plant species.

CRISPR载体的构建与DNA组装通常会产生数十到数百个独立的克隆。通过PCR克隆筛选容易识别正确的克隆，可以带和不带刀片（ 图2A），这是用于故障排除质粒进行区分。通常情况下，所有的克隆包含插件和用户可以选择完全跳过克隆筛选步骤。诊断消化（ 图2B）和Sanger测序被用于质量控制。什么时候发生错误，它们通常在重叠DNA区域观察到的， ...

Watch this Scientific Journal Video about High-throughput CRISPR Vector Construction and Characterization of DNA Modifications by Generation of Tomato Hairy Roots at JoVE.com

High-throughput CRISPR Vector Construction and Characterization of DNA Modifications by Generation of Tomato Hairy Roots

高通量CRISPR载体的构建及DNA修饰的表征番茄毛状根的一代

使用DNA装配，可以并行地构建在单个克隆反应多个CRISPR矢量，使得大量CRISPR的建设矢量a简单的任务。番茄毛状根是一个很好的模型系统来验证CRISPR载体和产生突变体材料。

Targeted DNA mutations generated by vectors with clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology have proven useful for ...

high-throughput-crispr-vector-construction-characterization-dna

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

17.8K 次观看.  Boyce Thompson Institute for Plant Research. 使用DNA装配，可以并行地构建在单个克隆反应多个CRISPR矢量，使得大量CRISPR的建设矢量a简单的任务。番茄毛状根是一个很好的模型系统来验证CRISPR载体和产生突变体材料。 

视频: High-throughput CRISPR Vector Construction and Characterization of DNA Modifications by Generation of Tomato Hairy Roots

Transient Expression in Nicotiana Benthamiana Leaves for Triterpene Production at a Preparative Scale

The triterpenes are one of the largest and most structurally diverse families of plant natural products. Many triterpene derivatives have been shown to possess medicinally relevant biological activity. However, thus far this potential has not translated into a plethora of triterpene-derived drugs in the clinic. This is arguably (at least partially) a consequence of limited practical synthetic access to this class of compound, a problem that can stifle the exploration of structure-activity relationships and development of lead candidates by traditional medicinal chemistry workflows. Despite their immense diversity, triterpenes are all derived from a single linear precursor, 2,3-oxidosqualene. Transient heterologous expression of biosynthetic enzymes in N. benthamiana can divert endogenous supplies of 2,3-oxidosqualene towards the production of new high-value triterpene products that are not naturally produced by this host. Agro-infiltration is an efficient and simple means of achieving transient expression in N. benthamiana. The process involves infiltration of plant leaves with a suspension of Agrobacterium tumefaciens carrying the expression construct(s) of interest. Co-infiltration of an additional A. tumefaciens strain carrying an expression construct encoding an enzyme that boosts precursor supply significantly increases yields. After a period of five days, the infiltrated leaf material can be harvested and processed to extract and isolate the resulting triterpene product(s). This is a process that is linearly and reliably scalable, simply by increasing the number of plants used in the experiment. Herein is described a protocol for rapid preparative-scale production of triterpenes utilizing this plant-based platform. The protocol utilizes an easily replicable vacuum infiltration apparatus, which allows the simultaneous infiltration of up to four plants, enabling batch-wise infiltration of hundreds of plants in a short period of time.

Transient Expression in Nicotiana Benthamiana Leaves for Triterpene Production at a Preparative Scale

Transient heterologous expression of biosynthetic enzymes in Nicotiana benthamiana leaves can divert endogenous supplies of 2,3-oxidosqualene towards the production of new high-value triterpene products. Herein is described a detailed protocol for rapid (5 days) preparative-scale production of triterpenes and analogs utilizing this powerful plant-based platform.

烟草 Benthamiana叶在制备鳞片上的瞬态表达

The triterpenes are one of the largest and most structurally diverse families of plant natural products. Many triterpene derivatives have been shown to ...

科研

生物化学

Tomato Root Transformation Followed by Inoculation with Ralstonia Solanacearum for Straightforward Genetic Analysis of Bacterial Wilt Disease

Ralstonia solanacearum is a devastating soil borne vascular pathogen that can infect a large range of plant species, causing an important threat to agriculture. However, the Ralstonia model is considerably underexplored in comparison to other models involving bacterial plant pathogens, such as Pseudomonas syringae in Arabidopsis. Research targeted to understanding the interaction between Ralstonia and crop plants is essential to develop sustainable solutions to fight against bacterial wilt disease but is currently hindered by the lack of straightforward experimental assays to characterize the different components of the interaction in native host plants. In this scenario, we have developed a method to perform genetic analysis of Ralstonia infection of tomato, a natural host of Ralstonia. This method is based on Agrobacterium rhizogenes-mediated transformation of tomato roots, followed by Ralstonia soil-drenching inoculation of the resulting plants, containing transformed roots expressing the construct of interest. The versatility of the root transformation assay allows performing either gene overexpression or gene silencing mediated by RNAi. As a proof of concept, we used this method to show that RNAi-mediated silencing of SlCESA6 in tomato roots conferred resistance to Ralstonia. Here, we describe this method in detail, enabling genetic approaches to understand bacterial wilt disease in a relatively short time and with small requirements of equipment and plant growth space.

Tomato Root Transformation Followed by Inoculation with Ralstonia Solanacearum for Straightforward Genetic Analysis of Bacterial Wilt Disease

Here, we present a versatile method for tomato root transformation followed by inoculation with Ralstonia solanacearum to perform straightforward genetic analysis for the study of bacterial wilt disease.

番茄根转化后与拉子托尼亚索拉纳卡鲁姆接种，直接遗传分析细菌性威尔特病

Ralstonia solanacearum is a devastating soil borne vascular pathogen that can infect a large range of plant species, causing an important threat to ...

遗传学

Inducing Hairy Roots by Agrobacterium rhizogenes-Mediated Transformation in Tartary Buckwheat (Fagopyrum tataricum)

Tartary buckwheat (TB) [Fagopyrum tataricum (L.) Gaertn] possesses various biological and pharmacological activities because it contains abundant secondary metabolites such as flavonoids, especially rutin. Agrobacterium rhizogenes have been gradually used worldwide to induce hairy roots in medicinal plants to investigate gene functions and increase the yield of secondary metabolites. In this study, we have described a detailed method to generate A. rhizogenes-mediated hairy roots in TB. Cotyledons and hypocotyledonary axis at 7&#8211;10 days were selected as explants and infected with A. rhizogenes carrying a binary vector, which induced adventitious hairy roots that appeared after 1 week. The generated hairy root transformation was identified based on morphology, resistance selection (kanamycin), and reporter gene expression (green fluorescent protein). Subsequently, the transformed hairy roots were self-propagated as required. Meanwhile, a myeloblastosis (MYB) transcription factor, FtMYB116, was transformed into the TB genome using the A. rhizogenes-mediated hairy roots to verify the role of FtMYB116 in synthesizing flavonoids. The results showed that the expression of flavonoid-related genes and the yield of flavonoid compounds (rutin and quercetin) were significantly (p &#60; 0.01) promoted by FtMYB116, indicating that A. rhizogenes-mediated hairy roots can be used as an effective alternative tool to investigate gene functions and the production of secondary metabolites. The detailed step-by-step protocol described in this study for generating hairy roots can be adopted for any genetic transformation or other medicinal plants after adjustment.

Inducing Hairy Roots by Agrobacterium rhizogenes-Mediated Transformation in Tartary Buckwheat Fagopyrum tataricum

We describe a method of inducing hairy roots by Agrobacterium rhizogenes-mediated transformation in Tartary buckwheat (Fagopyrum tataricum). This can be used to investigate gene functions and production of secondary metabolites in Tartary buckwheat, be adopted for any genetic transformation, or used for other medicinal plants after improvement.

诱导毛茸茸的根由农业细菌根- 在塔特里巴克麦的中介转化 （法戈皮鲁姆塔塔里库姆）

Tartary buckwheat (TB) [Fagopyrum tataricum (L.) Gaertn] possesses various biological and pharmacological activities because it contains abundant ...

CcCIPK14 Gene Function Analysis to Illuminate the Efficient Root Transgenic System

An efficient and stable transformation system is fundamental for gene function study and molecular breeding of plants. Here, we describe the use of an Agrobacterium rhizogenes mediated transformation system on pigeon pea. The stem is infected with A. rhizogenes carrying a binary vector, which induced callus after 7 days and adventitious roots 14 days later. The generated transgenic hairy root was identified by morphological analysis and a GFP reporter gene.To further illustrate the application range of this system, CcCIPK14 (Calcineurin B-like protein-interacting protein kinases) was transformed into pigeon pea using this transformation method. The transgenic plants were treated with jasmonic acid (JA) and abscisic acid (ABA), respectively, for the purpose of testing whether CcCIPK14 responds to those hormones. The results demonstrated that (1) exogenous hormones could significantly upregulate the expression levelof CcCIPK14, especially in CcCIPK14 over-expression (OE) plants; (2) the content of Genistein in CcCIPK14-OE lines was significantly higher than the control; (3) the expression level of two downstream key flavonoid synthase genes, CcHIDH1 and CcHIDH2, were up-regulated in the CcCIPK14-OE lines; and (4) the hairy root transgenic system can be used to study metabolically functional genes in non-model plants.

CcCIPK14 Gene Function Analysis to Illuminate the Efficient Root Transgenic System

Here we present an efficient and stable transformation system for the functional analysis of the CcCIPK14 gene as an example, providing a technical basis for studying the metabolism of non-model plants.

An efficient and stable transformation system is fundamental for gene function study and molecular breeding of plants. Here, we describe the use of an ...

使用 CRISPRmass 的高通量 UAS-cDNA/ORF 质粒文库构建

CRISPR-Based Modular Assembly for High-Throughput Construction of a UAS-cDNA/ORF Plasmid Library

Functional genomics screening offers a powerful approach to probe gene function and relies on the construction of genome-wide plasmid libraries. Conventional approaches for plasmid library construction are time-consuming and laborious. Therefore, we recently developed a simple and efficient method, CRISPR-based modular assembly (CRISPRmass), for high-throughput construction of a genome-wide upstream activating sequence-complementary DNA/open reading frame (UAS-cDNA/ORF) plasmid library. Here, we present a protocol for CRISPRmass, taking as an example the construction of a GAL4/UAS-based UAS-cDNA/ORF plasmid library. The protocol includes massively parallel two-step test tube reactions followed by bacterial transformation. The first step is to linearize the existing complementary DNA (cDNA) or open reading frame (ORF) cDNA or ORF library plasmids by cutting the shared upstream vector sequences adjacent to the 5&#39; end of cDNAs or ORFs using CRISPR/Cas9 together with single guide RNA (sgRNA), and the second step is to insert a UAS module into the linearized cDNA or ORF plasmids using a single step reaction. CRISPRmass allows the simple, fast, efficient, and cost-effective construction of various plasmid libraries. The UAS-cDNA/ORF plasmid library can be utilized for gain-of-function screening in cultured cells and for constructing a genome-wide transgenic UAS-cDNA/ORF library in Drosophila.&#160;

作者聚焦:使用 CRISPRmass 简化全基因组质粒文库构建

We present a protocol for CRISPR-based modular assembly (CRISPRmass), a method for high-throughput construction of UAS-cDNA/ORF plasmid library in Drosophila using publicly available cDNA/ORF resources. CRISPRmass can be applied to editing various plasmid libraries.

基于CRISPR的模块化组装用于UAS-cDNA/ORF质粒文库的高通量构建

Functional genomics screening offers a powerful approach to probe gene function and relies on the construction of genome-wide plasmid libraries. ...

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.

使用模块化金门克隆的多基因结构快速组装

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

生物工程

转基因大豆毛状根的高效生产

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.

作者聚焦:用于基因功能分析的大豆毛状根转化  

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

大豆毛根转化基因功能分析

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

高通量CRISPR载体的构建及DNA修饰的表征番茄毛状根的一代

摘要

探索更多视频

此视频中的章节

烟草 Benthamiana叶在制备鳞片上的瞬态表达

番茄根转化后与拉子托尼亚索拉纳卡鲁姆接种，直接遗传分析细菌性威尔特病

诱导毛茸茸的根由农业细菌根- 在塔特里巴克麦的中介转化（法戈皮鲁姆塔塔里库姆）

CcCIPK14 Gene Function Analysis to Illuminate the Efficient Root Transgenic System

基于CRISPR的模块化组装用于UAS-cDNA/ORF质粒文库的高通量构建

基于CRISPR的模块化组装用于UAS-cDNA/ORF质粒文库的高通量构建

基于CRISPR的模块化组装用于UAS-cDNA/ORF质粒文库的高通量构建

使用模块化金门克隆的多基因结构快速组装

大豆毛根转化基因功能分析

大豆毛根转化基因功能分析

高通量CRISPR载体的构建及DNA修饰的表征番茄毛状根的一代

摘要

探索更多视频

此视频中的章节

烟草 Benthamiana叶在制备鳞片上的瞬态表达

番茄根转化后与拉子托尼亚索拉纳卡鲁姆接种，直接遗传分析细菌性威尔特病

诱导毛茸茸的根由农业细菌根- 在塔特里巴克麦的中介转化 （法戈皮鲁姆塔塔里库姆）

CcCIPK14 Gene Function Analysis to Illuminate the Efficient Root Transgenic System

基于CRISPR的模块化组装用于UAS-cDNA/ORF质粒文库的高通量构建

基于CRISPR的模块化组装用于UAS-cDNA/ORF质粒文库的高通量构建

基于CRISPR的模块化组装用于UAS-cDNA/ORF质粒文库的高通量构建

使用模块化金门克隆的多基因结构快速组装

大豆毛根转化基因功能分析

大豆毛根转化基因功能分析

诱导毛茸茸的根由农业细菌根- 在塔特里巴克麦的中介转化（法戈皮鲁姆塔塔里库姆）