作者感谢以下本科生和高中生在镰刀菌突变体中产生OSCAR突变体:Anjellica Miller，Athar Naseer，Xiu Lin，Katelyn Woodburry，Chelsea Patterson，Kathleen Robertson，Krystina Bradley，Ashton Rogers，Alexis McKensie，Manny Hernandez ，Ashli​​ Crepsac，Jeff Delong，Christian King，Je Jeong，Maria Belding，Christy Burre，Daniel O&#39;Meara，Lauren（Victoria）Cook，Jake Goodman，Sampriti De，Oge Okoye，Alyssa Beckstead，Garrett Hibbs，Nick Goldstein，Caroline Twum ，Chris Benson，Louis Stokes，Hannah Itell，Jane Hulse，Jasim Mohammed，James Loggins，Kelli Russell，Gre&#39;Nisha Jones，Kristin Sheaffer，Mariam Hammady，Ava Wilson，Katrina Bazemore，Toney Harper，Karlin McGhee，Mohmed Momin，Rima Momin ，Thi Ngoc Le和Angel Pham。

1.基因片段的PCR扩增引物设计<ol><li>下载到文字处理文件中，包含开放阅读框（ORF）的目标基因（GOI）的基因组区域和来自真核生物数据库或其他基因组数据资源的每一侧的基因侧翼至少2 kb。 </li><li>突出显示要删除的ORF，并标记起始和终止密码。 </li><li>在下载的序列中识别并突出显示相邻ORF。 </li><li> 使用GOI ORF的2 kb 5&#39;端和引物设计工具（参见材料列表）设计PCR引物对O1和O2，以产生1 kb的最小大小的产物。小心不要影响相邻的ORF。 <ol><li>将2 kb 5&#39;侧面粘贴到"序列条目"窗口中。单击"显示自定义参数"框。输入以下定制设计参数:底漆TM 58（最小），60（opt）和62（最大）;底漆GC 40（min），50（opt）和60（max）;底漆尺寸22（min），24（opt）和26（最大）;放大镜尺寸1250（min），1500（opt）和2000（最大）。 </li><li>选择反向引物（O2）最接近ORF的结果。捕获测定的屏幕截图，并将引物序列复制并粘贴到文字处理文档中。 </li><li>重复3&#39;侧翼的过程以产生引物O3和O4，此时选择将最接近目标ORF的正向引物（O3）放置的结果。 </li></ol></li><li>在引物1至4的5&#39;末端添加适当的连接位点（ 参见表1 ）。 </li><li>还可以在下面的第4部分中设计和订购用于删除确认的ORF特异性引物。参数应与步骤1.4.1中的相同，但必要时使用Amplicon Size 500（min），750（opt）和1000（max）调整ORF的长度。最后，为了确认同源重组体，产生位于染色体外侧的外侧的5&#39;端和3&#39;端引物。这些"出"引物将与之配合使用hygR（210）和hygF（850）（ 图2 ）。 </li><li>订购底漆。 </li></ol> 2.生产OSCAR构造<ol><li>使用高保真Taq，在一个反应​​中使用引物（100μM）O1和O2，在第二次引物O3和O4中进行两个反应，每个用于5&#39;和3&#39;侧翼。反应混合物含有:29.5μL无菌蒸馏水（SDW），5μL10x LA PCR缓冲液，8μL2.5mM dNTP混合物，5μL25mM MgCl 2，1μL模板（50ng /μL），0.5μLhi-保真度Taq ，0.5μLPrimer O1和0.5μL5&#39;侧翼的引物O2（或0.5μL引物O3和0.5μL引物O4用于3&#39;侧翼）使用反应条件如下:94℃1分钟，然后30个循环94℃30秒，60℃30秒，72℃2分钟，然后最终步骤72℃5分钟，然后无限期地保持在10℃。 </li><li>运行0.8％琼脂糖1×TAE（40mM Tris乙酸盐pH8.3,1mM EDTA）凝胶电泳，在标准微型凝胶装置中约125Vh，以测定产物的大小和相对浓度。根据制造商的说明书，用非乙腈染色将凝胶染色。在紫外线照明器上可视化。 </li><li>如果5&#39;和3&#39;侧面产物大致均匀浓度，将它们组合并使用亲和柱试剂盒（或PEG沉淀90μL组合PCR产物，240μLTE缓冲液pH7.5,160μL30％聚乙二醇PEG8000 30mM MgCl 2 ）根据制造商的方案共同纯化PCR产物。如果它们不是相同的浓度，则所有的低浓度产物与来自较高产量反应的估计等量的产物结合。 </li><li>使用分光光度计测量纯化的DNA浓度。 </li><li>通过混合以下物质进行BP反应:将1μL的60ng /μLpOSCAR，2μL的60ng /μLpA-Hyg-OSCAR，1μL60 ng /μL组合侧腹，1μL克隆酶。在25°C孵育16 h。通过加入0.5μL蛋白酶K终止反应，并在37℃下孵育10分钟。 </li><li>改变高素质大肠杆菌细胞成功地使用市售的感受态细胞和自制的DH5αCaCl 2感受态细胞作为接受者，如制造商的说明书所述。在含有100μg/ mL壮观霉素（Spec）的低钠（0.5g / L NaCl）LB上。 </li><li>通过执行上面产生的菌落的DNA微量排泄10来鉴定正确的构建体。用Hin dIII和Kpn I进行双重消化 ，如下:移液5μLDNA，2 U酶，2μL适当的10倍缓冲液，SDW至20μL总体积。这从载体（ 约 7kb）释放插入片段（约1.5kb的1.5kb基因侧翼）并切割其中的任何位点侧面可以提供可预测的带尺寸。 </li><li>序列11选择显示适当条带图案的克隆。 </li></ol> 3. 农杆菌介导的真菌转化（ATMT） <ol><li>用OSCAR缺失构建体转化根癌土壤杆菌菌株AGL1。 </li><li> 用含有GOI OSCAR缺失质粒的AGL1转化真菌。要执行以下步骤: <ol><li>用2×10 6孢子/ mL的无菌水制备真菌孢子悬浮液。在血细胞计数器或自动细胞计数器上量化孢子。将其放入1.5 mL微量离心管中。这个设置是4个转换板。 </li><li>使用蓝色的无菌一次性回路，从2-3天龄的LB-Spec 100培养基中添加容易看到的含有AGL1的缺失质粒（应覆盖环直径约20％）的糖（见Maateials列表的组成）含有板并混合到孢子悬浮液中。漩涡直到细菌细胞分散（ 约 5分钟中等速度）。 </li><li>将100μL的分生孢子 - Agro悬浮液移至放置在含有共培养培养基12的四个6cm培养皿中的每一个上的纤维素膜过滤器的中心。 </li><li>用无菌玻璃珠（约4）涂覆悬浮液以覆盖膜过滤器的整个表面。或者使用传统的吊具工具分散悬架。将膜过滤器在罩中干燥约10分钟，用石蜡包裹。重复步骤3.2.2和3.2.3设置多个转换。 </li><li>在室温下孵育板2天，倒置。 </li><li>将膜过滤器转移到含有潮霉素B（Hyg）150μg/ mL，200mM头孢噻肟和100μg/ mL的6cm Aspergillus选择培养基拉氧头孢。在室温下孵育5-7天，然后分离Hyg抗性菌落。 </li><li>用无菌牙签将推定的转化子转移到含有150μg/ mL Hyg，100μg/ mL卡那霉素的6cm土豆葡萄糖琼脂（PDA）板上。 </li></ol></li></ol> 4.通过PCR删除突变体鉴定<ol><li> 通过热解从转化体中提取DNA进行PCR。 <ol><li>一旦转化体在步骤3.2.7的PDA上形成菌落，使用无菌牙签将少量（2×2mm）的菌丝体或酵母细胞从菌落转移到100μL裂解溶液（50mM磷酸钠，pH 7.4,1mM EDTA，5％甘油）中。 </li><li>在85-90℃下孵育混合物20-30分钟。将含有基因组DNA的粗提取物储存在-20℃直到在步骤4.2中使用。 </li></ol></li><li>对每个转化体进行4次PCR反应，每次转染d分别检测5&#39;和3&#39;侧翼的同源重组，一个用于可选择标记（在这种情况下为潮霉素磷酸转移酶），一个用于GOI ORF。 注意:我们通常使用廉价的低保真Taq进行这些反应。反应条件如上述步骤2.1所述，含有SDW20μL，10×Taq缓冲液2.5μL，dNTPS（10mM）0.5μL，自制Taq14,0.5μL，引物A（100μM）0.5μL，引物B（100μM） ）0.5μL，模板0.5μL。较低浓度的引物（10μM）可以很好地使用。 </li><li>运行PCR产物的琼脂糖凝胶（ 例如 0.8％）。删除突变体将产生Hyg带，但不产生ORF产物。异位整合将显示两条带。野生型只会产生ORF带。 </li><li>永久存储删除突变体（和异位转化子或两个用于控件）以备将来使用。 注意:使用15％甘油长期在-80°C下撕裂我们的菌株。 </li></ol>

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作者没有什么可以披露的。

已经成功地应用了农杆菌的一步构建 - 组合就绪质粒（OSCAR），其中越来越多的子囊菌真菌。假设农杆菌介导的转化和同源重组是可能的，该方法还应该容易地应用于担子菌门和其他真菌门（具有适当的启动子驱动选择性标记基因）的物种。已经产生了额外的标记载体以使抗真菌化合物的选择多样化，并且允许产生双重和高级突变体。这些包括抗G418，奈瑟菌素，最近除草剂草铵膦和氯嘧磺隆。 该方法用于大丽花黄堇，这里提供的图像主要来源于1 。最近OSCAR被广泛用于删除的真菌致病性真菌Fusarium v​​erticillioides中的基因。已经制作了超过60个缺失构建体，并且产生了超过30个基因的缺失突变体（未发表）。 在目前的迭代过程中，需要大约4-6周的时间来确定一组确认的缺失突变体。以下是主要OSCAR步骤和完成它们所需的大概时间的简要列表:1）基于基因组数据（5天）设计和采购OSCAR引物，2）侧翼扩增和克隆，（2天），3）通过微量制备和测序（1周）进行克隆确认，4）将质粒导入农杆菌进行ATMT（4天），5）ATMT在镰刀菌中产生转化体并生长（2周，注意到这取决于生长速率的真菌），热解和PCR测定转化体基因型（2天），6）单次打浆，基因型确认和储存（1-2周）。 <p cl屁股="jove_content"&gt; OSCAR对真正的高吞吐量的适应性并没有被我们所知，而应该是实用的。这些反应足够坚固，以便成功应用于多孔格式。机器人的使用肯定会推动这种方法。 OSCAR方法具有需要包含扩增侧翼的单个重组反应和两种质粒pOSCAR和pA-Hyg OSCAR的时间优势。类似的方法需要产生必须确认的进入克隆，然后在第二反应中使用以产生感兴趣的构建体。提及的质粒pOSCAR和pA-Hyg OSCAR可通过Addgene或Fungal Genetics Stock Center获得。其他OSCAR标记质粒如pA-Bar-OSCAR和pA-Sur-OSCAR可以从作者5获得 。

遗传解剖是确定个体或基因组合的功能重要性的有力方法。了解特定基因作用的标准方法是在任何其他基因中不改变单基因突变体的产生。最强大和最不可能的混杂方法是完全和精确地删除感兴趣的基因开放阅读框架（GOI ORF）而不损害任何其他基因功能。 因为用于缺失质粒生成的标准连接方法需要多个步骤，OSCAR 1的合理性就是产生更快速的体外方法。 图1描述了OSCAR方法中的组装过程。本文描述的方法具有将单个多部分反应中的单个基因缺失载体的快速构建与随后的根癌土壤杆菌介导转录（ATMT）。 OSCAR非常快速，并且与其他策略（例如在酵母2中使用吉布森装配）相比较。 OSCAR方法成功应用于几种真菌菌根霉菌。这些物种包括: 镰孢镰孢 （未发表）， 大丽花 ， e 3 ，，ica ica ica ica ii ii ii ii ii ii um um um um um。。。。。 SP。 Vasinfectum 6 ，Pestalotiopsis microspora 7 ，Colletotrichum higginsianum 8 和Dothistroma septosporum 9和Sarocladium zeae （未发表） 。 该方案提供了包括引物设计，侧翼PCR扩增，OSCAR BP反应，缺失构建结构确认，转化的方法的逐步说明土壤杆菌的离子与构建物接着基于ATMT将缺失构建体转移到真菌细胞中，最后将真菌缺失突变体与具有异位整合的缺失构建体的突变体区分开。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>FungiDB</td>
			<td></td>
			<td></td>
			<td>Database/ http://fungidb.org/fungidb/</td>
		</tr>
		<tr>
			<td>IDT PrimerQuest</td>
			<td>IDT</td>
			<td></td>
			<td>Primer design online software/ http://www.idtdna.com/Primerquest/Home/Index</td>
		</tr>
		<tr>
			<td>Microsoft Word</td>
			<td></td>
			<td></td>
			<td>Sequence file manipulation</td>
		</tr>
		<tr>
			<td>Low Na LB Spec 100 medium</td>
			<td></td>
			<td></td>
			<td>E. coli transformant selection, composition: 1% tryptone, 0.05% NaCl, 0.5% yeast extract, 1.5 % agar if for solid medium</td>
		</tr>
		<tr>
			<td>Co-cultivation medium</td>
			<td></td>
			<td></td>
			<td>ATMT transformation induction (Reference 12)</td>
		</tr>
		<tr>
			<td>Aspergillus minimal medium with Hygromycin</td>
			<td></td>
			<td></td>
			<td>Fungal transformant selection</td>
		</tr>
		<tr>
			<td>PDA medium</td>
			<td>Acumedia</td>
			<td>7149A</td>
			<td>Single spore slant tubes</td>
		</tr>
		<tr>
			<td>PDA-Hyg-Kan medium</td>
			<td></td>
			<td></td>
			<td>Fungal ransformant isolation, PDA containing 150 &#956;g/ml hygromycin B and 100 &#956;g/ml Kanamycin;</td>
		</tr>
		<tr>
			<td>Glass beads</td>
			<td>Genlantis</td>
			<td>C400100</td>
			<td>Plate spreading</td>
		</tr>
		<tr>
			<td>Nitrocellulose filters (47mm)</td>
			<td>Fisher</td>
			<td>09-719-555</td>
			<td>Co-culturing for ATMT</td>
		</tr>
		<tr>
			<td>Various centifuge tubes</td>
			<td></td>
			<td></td>
			<td>multiple preps</td>
		</tr>
		<tr>
			<td>Petri plates (various)</td>
			<td></td>
			<td></td>
			<td>Culturing of bacteria and Fungi</td>
		</tr>
		<tr>
			<td>pA-Hyg OSCAR</td>
			<td>Addgene</td>
			<td>29640</td>
			<td>Selectable marker vector</td>
		</tr>
		<tr>
			<td>pOSCAR</td>
			<td>Addgene</td>
			<td>29639</td>
			<td>Assembly vector</td>
		</tr>
		<tr>
			<td>DH5a One Shot Competent E. coli cells</td>
			<td>Life Technologies&#160;</td>
			<td>12297-016</td>
			<td>BP reaction transformation</td>
		</tr>
		<tr>
			<td>ccdB survival E. coli cells</td>
			<td>Life Technologies&#160;</td>
			<td>A10460</td>
			<td>Maintenance of pOSCAR</td>
		</tr>
		<tr>
			<td>Wooden transfer sticks</td>
			<td></td>
			<td></td>
			<td>Colony streaking</td>
		</tr>
		<tr>
			<td>Toothpicks</td>
			<td></td>
			<td></td>
			<td>Colony picking</td>
		</tr>
		<tr>
			<td>Microcentrifuge</td>
			<td></td>
			<td></td>
			<td>Pelleting Bacteria etc</td>
		</tr>
		<tr>
			<td>Preparative centrifuge</td>
			<td></td>
			<td></td>
			<td>Fungal spore collection</td>
		</tr>
		<tr>
			<td>Dissecting microscope</td>
			<td></td>
			<td></td>
			<td>Single spore isolation</td>
		</tr>
		<tr>
			<td>Automated Cell Counter</td>
			<td></td>
			<td></td>
			<td>Spore suspension calculation</td>
		</tr>
		<tr>
			<td>Compound microscope</td>
			<td></td>
			<td></td>
			<td>Hemocytometer cell counting</td>
		</tr>
		<tr>
			<td>QIAquick PCR Purification Kit&#160;</td>
			<td>Qiagen</td>
			<td>28104</td>
			<td>PCR gene flank produict purification</td>
		</tr>
		<tr>
			<td>TaKaRa LA Taq&#160;</td>
			<td>Takara Bio USA</td>
			<td>RR002A</td>
			<td>Hi Fidelity taq polymerase for OSCAR flank generation</td>
		</tr>
		<tr>
			<td>Hygromycin B</td>
			<td>InvivoGen</td>
			<td>ant-hg-5</td>
			<td></td>
		</tr>
		<tr>
			<td>Spectinomycin</td>
			<td>Sigma</td>
			<td>22189-32-8</td>
			<td></td>
		</tr>
		<tr>
			<td>Cefotaxim&#160;</td>
			<td>TCI America</td>
			<td>C2224</td>
			<td></td>
		</tr>
		<tr>
			<td>Moxalactam&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>43963</td>
			<td></td>
		</tr>
		<tr>
			<td>GelRed</td>
			<td>&#160;Phenix Research Products</td>
			<td>RGB-4103</td>
			<td>Post staining agarose gels</td>
		</tr>
		<tr>
			<td>Qiagen QIAquick PCR Purification Kit (Cat. No. 28104)&#160;</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>(OneShot_ Mach1TM T1R or One Shot_ OmniMAX&#8482; 2 T1R from Invitrogen)&#160;</td>
			<td>Thermo Fisher Scientific</td>
			<td>C862003</td>
			<td></td>
		</tr>
		<tr>
			<td>Gateway BP Clonase II Enzyme mix</td>
			<td>Thermo Fisher Scientific</td>
			<td>11789020</td>
			<td>Used to assemble deletion construct in pOSAR</td>
		</tr>
		<tr>
			<td>PrimerQuest tool</td>
			<td>IDT&#160;</td>
			<td></td>
			<td>Used in step 1.4; available on http://www.idtdna.com/Primerquest/Home/Index</td>
		</tr>
	</tbody>
</table>

rapid deletion production in fungi via agrobacterium mediated transformation of oscar deletion constructs

精确删除感兴趣的基因，同时使基因组的其余部分保持不变，为确定特定基因在活体中的功能提供了理想的产物。在本协议中，描述了精确快速删除质粒构建的OSCAR方法。 OSCAR依赖于进行单次重组酶反应的克隆系统，其中包含纯化的PCR扩增的目的基因的5&#39;和3&#39;侧翼和两个质粒pA-Hyg OSCAR（标记载体）和pOSCAR（组装体向量）。正确组装的缺失载体的确认通过限制性消化作图进行测序。然后使用根癌土壤杆菌介导将缺失构建体引入真菌孢子（简称ATMT）。最后，描述PCR测定以确定是否通过同源或非同源重组整合的缺失构建体，表明基因缺失或异位整合。这种方法已被成功地用于删除大丽花轮枝 菌属和其他物种中的尖角 镰孢属（Fusarium v​​erticillioides）中的许多基因。

在单个反应中，OSCAR方法产生含有围绕选择性标记盒的待缺失的靶基因的侧翼的质粒。使用OSCAR生成删除结构非常有效。然而，该系统可以产生含有一些但不是全部三个片段（两个基因侧翼和可选择标记）的部分构建体。通常，大多数大肠杆菌转化体含有正确的OSCAR构建体。例如， 图2描述了大丽轮枝菌基因VDAG_06812的OSCAR缺失构建体。在这种情况下，VDAG_06812的侧翼缺少Hin dIII或KpnI位点，因此应释放出4,247 bp的质粒插入片段。 图3显示了正常质粒结构的Hin dIII- Kpn I限制酶双重消化确认，除了代表异常构建的泳道5和11TS。 图4显示了通过Southern印迹的两种不同基因缺失的最终确认。 图5显示了作为Southern印迹的替代方案的确定性PCR方法。 <img alt="图2" src="/files/ftp_upload/55239/55239fig1.jpg" /> 图1:OSCAR删除构建反应。 OSCAR缺失构建过程包括5&#39;和3&#39;基因侧翼的单独的PCR扩增，随后与组合的纯化的侧翼产物和二元选择标记质粒进行BP克隆酶反应。 B1r，B2r，B3，B4，P1r，P2r，P3，P4，R1，R2，L3和L4表示λ重组位点。重新打印参考1的许可。 <img alt="图2" src="/files/ftp_upload/55239/55239fig2.jpg" /> 图2: OSCAR删除结构<html> em&gt;大丽轮枝菌 基因VDAG_06812。显示限制酶识别和引物退火位点的位置以验证正确的缺失构建体结构。重新打印参考1的许可。 <img alt="图3" src="/files/ftp_upload/55239/55239fig3.jpg" /> 图3: 使用 Kpn I和 Hin dIII对VDAG_06812的缺失构建体结构进行限制性 消化 确认 。将质粒双重消化并通过在0.8％琼脂糖凝胶上的凝胶电泳进行分析。正确的构建体产生约6.9kb和4.2kb的两条带，分别表示与靶基因侧翼融合的二元载体骨架和HygR标记。重新打印参考1的许可。 ontent"fo:keep-together.within-page ="1"&gt; <img alt="图4" src="/files/ftp_upload/55239/55239fig4.jpg" /> 图4: 使用OSCAR缺失构建体 确认大丽花中VDAG_02161和VDAG_09930的Southern印迹杂交 。基因组DNA用Bcl I消化。然后用VDAG_02161和VDAG_09930 ORF探针同时检测它们。分别为VDAG_02161和VDAG_09930的野生型等位基因的杂交带分别为2,757 bp和1,426 bp。样品如下:泳道1:野生型菌株VdLs.17;泳道2:缺失突变菌株02161.1;泳道3:缺失突变菌株02161.3;泳道4:异位转化株Ect 02161.2;泳道5:缺失突变菌株09930.3;泳道6:缺失突变菌株09930.10;泳道7:异位转化株Ect 09930.4。通过许可重新打印 参考文献1 。 图5: 通过PCR的缺失突变体确认。分析基因FVEG_13253的缺失和异位镰刀菌轮枝菌转化体。泳道1顶部和底部，标记;泳道2-6顶部凝胶转化体PCR 5&#39;末端片段扩增;泳道7-11顶部凝胶ORF扩增;泳道2-6底部凝胶Hyg基因扩增;泳道7-11底部凝胶3&#39;出片段扩增。样品是缺失菌株11/31（泳道2和7），11/32（泳道3和8），11/33（泳道4和9）和异位转化体11/34（泳道5和10）和11 / 35（泳道6和11）。 <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td> 底漆 </td><td> 使用 </td><td> 序列 </td></tr><tr><td>引物O1-（attB2r） </td><td> 5&#39;侧翼扩增，引导向前</td><td> 5&#39;-GGGGACAGCTTTCTTGTACAAAGTGGAA </td></tr><tr><td>底漆O2-（attB1r） </td><td> 5&#39;侧翼扩增，引物反转</td><td> 5&#39;-GGGGACTGCTTTTTTGTACAAACTTGT </td></tr><tr><td>引物O3-（attB4） </td><td> 3&#39;侧翼扩增，引物向前</td><td> 5&#39;-GGGGACAACTTTGTATAGAAAAGTTGTT </td></tr><tr><td>引物O4-（attB3） </td><td> 3&#39;侧翼，引物反向扩增</td><td> 5&#39;-GGGGACAACTTTGTATAATAAAGTTGT </td></tr></tbody></table> 表1:加入基因特异性引物序列的特异性OSCAR引物5&#39;延伸。 <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td> 底漆 </td><td> 使用 </td><td style="width:250px"> 序列 </td></tr><tr><td> OSC-F </td><td&gt; OSCAR正向测序引物，5&#39;侧翼的序列有义链</td><td> 5&#39;-CTAGAGGCGCGCCGATATCCT </td></tr><tr><td> OSC-R </td><td> OSCAR反向测序引物，5&#39;侧翼的序列反义链</td><td> 5&#39;-CGCCAATATATCCTGTCAAACACT </td></tr><tr><td> HygR（210） </td><td>反向测序引物，5&#39;侧翼的序列反义链</td><td> 5&#39;-GCCGATGCAAAGTGCCGATAAACA </td></tr><tr><td> HygF（850） </td><td>正向测序引物，3&#39;侧翼的序列有义链</td><td> 5&#39;-AGAGCTTGGTTGACGGCAATTTCG </td></tr><tr><td>新Hyg Marker前进</td><td>为了扩增潮霉素抗性基因作为对照（与下面的新的Hyg Marker反向引物一起） </td><td> 5&#39;-GACAGGAACGAGGACATTATTA </td></tr><tr><td>新的Hyg标记反向</td><td>将潮霉素抗性基因扩增为对照（连同上述新的Hyg Marker Forward引物）） </td><td> 5&#39;-GCTCTGATAGAGTTGGTCAAG </td></tr></tbody></table> 表2:用于分析OSCAR缺失构建体的引物。

Watch this Scientific Journal Video about Rapid Deletion Production in Fungi via Agrobacterium Mediated Transformation of OSCAR Deletion Constructs at JoVE.com

Rapid Deletion Production in Fungi via Agrobacterium Mediated Transformation of OSCAR Deletion Constructs

通过同源重组产生的基因缺失突变体是基因功能研究的黄金标准。描述了用于快速产生缺失构建体的OSCAR（农杆菌 - 重组 - 质粒的一步构建）方法。 土壤杆菌介导的真菌转化如下。最后，提出了基于真菌转化体基因缺失的基于PCR的确认方法。

真菌快速缺失生产<em&gt;农杆菌</em&gt;介入OSCAR删除构造的转换

Precise deletion of gene(s) of interest, while leaving the rest of the genome unchanged, provides the ideal product to determine that particular ...

rapid-deletion-production-fungi-via-agrobacterium-mediated

Nanyang Technological University

Research

JoVE Journal

Genetics

Rapid Deletion Production in Fungi via Agrobacterium Mediated Transformation of OSCAR Deletion Constructs

9.9K 次观看.  NPRC, USDA-ARS. 通过同源重组产生的基因缺失突变体是基因功能研究的黄金标准。描述了用于快速产生缺失构建体的OSCAR（农杆菌 - 重组 - 质粒的一步构建）方法。 土壤杆菌介导的真菌转化如下。最后，提出了基于真菌转化体基因缺失的基于PCR的确认方法。 

视频: Rapid Deletion Production in Fungi via Agrobacterium Mediated Transformation of OSCAR Deletion Constructs

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.

机器人平台的高通量原生质体分离和转型

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 ...

科研

遗传学

Rearing and Double-stranded RNA-mediated Gene Knockdown in the Hide Beetle, Dermestes maculatus

Advances in genomics have raised the possibility of probing biodiversity at an unprecedented scale. However, sequence alone will not be informative without tools to study gene function. The development and sharing of detailed protocols for the establishment of new model systems in laboratories, and for tools to carry out functional studies, is thus crucial for leveraging the power of genomics. Coleoptera (beetles) are the largest clade of insects and occupy virtually all types of habitats on the planet. In addition to providing ideal models for fundamental research, studies of beetles can have impacts on pest control as they are often pests of households, agriculture, and food industries. Detailed protocols for rearing and maintenance of D. maculatus laboratory colonies and for carrying out dsRNA-mediated interference in D. maculatus are presented. Both embryonic and parental RNAi procedures-including apparatus set up, preparation, injection, and post-injection recovery-are described. Methods are also presented for analyzing embryonic phenotypes, including viability, patterning defects in hatched larvae, and cuticle preparations for unhatched larvae. These assays, together with in situ hybridization and immunostaining for molecular markers, make D. maculatus an accessible model system for basic and applied research. They further provide useful information for establishing procedures in other emerging insect model systems.

Rearing and Double-stranded RNA-mediated Gene Knockdown in the Hide Beetle, Dermestes maculatus

Here, we present protocols for rearing an intermediate-germ beetle, Dermestes maculatus (D. maculatus) in the lab. We also share protocols for embryonic and parental RNAi and methods for analyzing embryonic phenotypes to study gene function in this species.

饲养并在隐藏甲壳虫双链RNA介导的基因敲除，<em&gt;白腹皮蠹</em

Advances in genomics have raised the possibility of probing biodiversity at an unprecedented scale. However, sequence alone will not be informative ...

CRISPR/Cas9-mediated Targeted Integration In Vivo Using a Homology-mediated End Joining-based Strategy

As a promising genome editing platform, the CRISPR/Cas9 system has great potential for efficient genetic manipulation, especially for targeted integration of transgenes. However, due to the low efficiency of homologous recombination (HR) and various indel mutations of non-homologous end joining (NHEJ)-based strategies in non-dividing cells, in vivo genome editing remains a great challenge. Here, we describe a homology-mediated end joining (HMEJ)-based CRISPR/Cas9 system for efficient in vivo precise targeted integration. In this system, the targeted genome and the donor vector containing homology arms (~800 bp) flanked by single guide RNA (sgRNA) target sequences are cleaved by CRISPR/Cas9. This HMEJ-based strategy achieves efficient transgene integration in mouse zygotes, as well as in hepatocytes in vivo. Moreover, a HMEJ-based strategy offers an efficient approach for correction of fumarylacetoacetate hydrolase (Fah) mutation in the hepatocytes and rescues Fah-deficiency induced liver failure mice. Taken together, focusing on targeted integration, this HMEJ-based strategy provides a promising tool for a variety of applications, including generation of genetically modified animal models and targeted gene therapies.

CRISPR/Cas9-mediated Targeted Integration In Vivo Using a Homology-mediated End Joining-based Strategy

The clustered regularly interspaced short palindromic repeats/CRISPR associated protein 9 (CRISPR/Cas9) system provides a promising tool for genetic engineering, and opens up the possibility of targeted integration of transgenes. We describe a homology-mediated end joining (HMEJ)-based strategy for efficient DNA targeted integration in vivo and targeted gene therapies using CRISPR/Cas9.

CRISPR/Cas9-mediated 目标集成在体内使用基于同源介导的端接连接策略

As a promising genome editing platform, the CRISPR/Cas9 system has great potential for efficient genetic manipulation, especially for targeted integration ...

A Protocol for the Production of Integrase-deficient Lentiviral Vectors for CRISPR/Cas9-mediated Gene Knockout in Dividing Cells

Lentiviral vectors are an ideal choice for delivering gene-editing components to cells due to their capacity for stably transducing a broad range of cells and mediating high levels of gene expression. However, their ability to integrate into the host cell genome enhances the risk of insertional mutagenicity and thus raises safety concerns and limits their usage in clinical settings. Further, the persistent expression of gene-editing components delivered by these integration-competent lentiviral vectors (ICLVs) increases the probability of promiscuous gene targeting. As an alternative, a new generation of integrase-deficient lentiviral vectors (IDLVs) has been developed that addresses many of these concerns. Here the production protocol of a new and improved IDLV platform for CRISPR-mediated gene editing and list the steps involved in the purification and concentration of such vectors is described and their transduction and gene-editing efficiency using HEK-293T cells was demonstrated. This protocol is easily scalable and can be used to generate high titer IDLVs that are capable of transducing cells in vitro and in vivo. Moreover, this protocol can be easily adapted for the production of ICLVs.

We describe the production strategy of integrase-deficient lentiviral vectors (IDLVs) as vehicles for delivering CRISPR/Cas9 to cells. With an ability to mediate quick and robust gene editing in cells, IDLVs present a safer and equally effective vector platform for gene delivery compared to integrase-competent vectors.

整合酶缺陷慢病毒载体载体的制备及其在细胞 CRISPR/Cas9-mediated 基因敲除中的表达

Lentiviral vectors are an ideal choice for delivering gene-editing components to cells due to their capacity for stably transducing a broad range of cells ...

Adapting 3' Rapid Amplification of CDNA Ends to Map Transcripts in Cancer

Maturation of eukaryotic mRNAs involves 3&#39; end formation, which involves the addition of a poly(A) tail. In order to map the 3&#39; end of a gene, the traditional method of choice is 3&#39; rapid amplification of cDNA ends (3&#39; RACE). Protocols for 3&#39; RACE require the careful design and selection of nested primers within the 3&#39; untranslated region (3&#39; UTR) of the target gene of interest. However, with a few modifications the protocol can be used to include the entire 3&#39; UTR and sequences within the open reading frame (ORF), providing a more comprehensive picture of the relationship between the ORF and the 3&#39; UTR. This is in addition to identification of the polyadenylation signal (PAS), as well as the cleavage and polyadenylation site provided by conventional 3&#39; RACE. Expanded 3&#39; RACE can detect unusual 3&#39; UTRs, including gene fusions within the 3&#39; UTR, and the sequence information can be used to predict potential miRNA binding sites as well as AU rich destabilizing elements that may affect the stability of the transcript.

The two different 3&#39; rapid amplification of cDNA ends (3&#39; RACE) protocols described here make use of two different DNA polymerases to map sequences that include a segment of the open reading frame (ORF), the stop codon, and the entire 3&#39; UTR of a transcript using RNA obtained from different cancer cell lines.

3 ' 快速扩增 CDNA 末端, 以图记录癌症

Maturation of eukaryotic mRNAs involves 3&#39; end formation, which involves the addition of a poly(A) tail. In order to map the 3&#39; end of a gene, the ...

CRISPR-Mediated Reorganization of Chromatin Loop Structure

Recent studies have clearly shown that long-range, three-dimensional chromatin looping interactions play a significant role in the regulation of gene expression, but whether looping is responsible for or a result of alterations in gene expression is still unknown. Until recently, how chromatin looping affects the regulation of gene activity and cellular function has been relatively ambiguous, and limitations in existing methods to manipulate these structures prevented in-depth exploration of these interactions. To resolve this uncertainty, we engineered a method for selective and reversible chromatin loop re-organization using CRISPR-dCas9 (CLOuD9). The dynamism of the CLOuD9 system has been demonstrated by successful localization of CLOuD9 constructs to target genomic loci to modulate local chromatin conformation. Importantly, the ability to reverse the induced contact and restore the endogenous chromatin conformation has also been confirmed. Modulation of gene expression with this method establishes the capacity to regulate cellular gene expression and underscores the great potential for applications of this technology in creating stable de novo chromatin loops that markedly affect gene expression in the contexts of cancer and development.

Chromatin looping plays a significant role in gene regulation; however, there have been no technological advances that allow for selective and reversible modification of chromatin loops. Here we describe a powerful system for chromatin loop re-organization using CRISPR-dCas9 (CLOuD9), demonstrated to selectively and reversibly modulate gene expression at targeted loci.

CRISPR 的染色质环结构重组

Recent studies have clearly shown that long-range, three-dimensional chromatin looping interactions play a significant role in the regulation of gene ...

Lentiviral Mediated Production of Transgenic Mice: A Simple and Highly Efficient Method for Direct Study of Founders

For almost 40 years, pronuclear DNA injection represents the standard method to generate transgenic mice with random integration of transgenes. Such a routine procedure is widely utilized throughout the world and its main limitation resides in the poor efficacy of transgene integration, resulting in a low yield of founder animals. Only few percent of animals born after implantation of injected fertilized oocytes have integrated the transgene. In contrast, lentiviral vectors are powerful tools for integrative gene transfer and their use to transduce fertilized oocytes allows highly efficient production of founder transgenic mice with an average yield above 70%. Furthermore, any mouse strain can be used to produce transgenic animal and the penetrance of transgene expression is extremely high, above 80% with lentiviral mediated transgenesis compared to DNA microinjection. The size of the DNA fragment that can be cargo by the lentiviral vector is restricted to 10 kb and represents the major limitation of this method. Using a simple and easy to perform injection procedure beneath the zona pellucida of fertilized oocytes, more than 50 founder animals can be produced in a single session of microinjection. Such a method is highly adapted to perform, directly in founder animals, rapid gain and loss of function studies or to screen genomic DNA regions for their ability to control and regulate gene expression in vivo.

Here, we present a protocol to promote transgene integration and production of founder transgenic mice with high efficacy by a simple injection of a lentiviral vector in the perivitelline space of a fertilized oocyte.

慢病毒载体介导的转基因小鼠生产: 一种简单高效的创始人直接研究方法

For almost 40 years, pronuclear DNA injection represents the standard method to generate transgenic mice with random integration of transgenes. Such a ...

Plant Growth and Agrobacterium-mediated Floral-dip Transformation of the Extremophyte Schrenkiella parvula

Schrenkiella parvula is an extremophyte adapted to various abiotic stresses, including multiple ion toxicity stresses. Despite high-quality genomic resources available to study how plants adapt to environmental stresses, its value as a functional genomics model and tool has been limited by the lack of a feasible transformation system. In this protocol, we report how to generate stable transgenic S. parvula lines using an Agrobacterium-mediated floral-dip method. We modified the transformation protocol used for A. thaliana to account for unique traits of S. parvula, such as an indeterminate flowering habit and a high epicuticular wax content on leaves. Briefly, S. parvula seeds were stratified at 4 &#176;C for five days before planting. Plants were grown at a photoperiod of a 14 h light and 10 h dark and a 130 &#181;mol m-2s-1 light intensity, at 22 &#176;C to 24 &#176;C. Eight to nine week-old plants with multiple inflorescences were selected for transformation. These inflorescences were dipped in an infiltration solution of Agrobacterium tumefaciens GV3101 carrying the pMP90RK plasmid. We performed two rounds of flower dipping with an interval of three to four weeks to increase the transformation efficiency. The T1 seeds were collected and dried for four weeks in a container with desiccants before germination to screen for candidate transformed lines. Resistance to BASTA was used to screen T1 plants. We sprayed the BASTA solution three times with an interval of three days starting at two week-old plants to reduce false positives. A BASTA drop test was performed on surviving individual plants to identify true positive transformants. The transformation efficiency was 0.033%, yielding 3&#8211;4 transgenic plants per 10,000 T1 seeds propagated.

Plant Growth and Agrobacterium-mediated Floral-dip Transformation of the Extremophyte Schrenkiella parvula

Agrobacterium-mediated transformation using a floral-dip method can be successfully employed to create stable transgenic lines of the extremophyte model Schrenkiella parvula. We present a protocol modified from that for Arabidopsis thaliana, considering different growth habits and physiological characteristics of the extremophyte.

植物生长与农杆菌介导的植物浸水转化

Schrenkiella parvula is an extremophyte adapted to various abiotic stresses, including multiple ion toxicity stresses. Despite high-quality genomic ...

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 ...

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.

真菌快速缺失生产

摘要

探索更多视频

此视频中的章节

机器人平台的高通量原生质体分离和转型

饲养并在隐藏甲壳虫双链RNA介导的基因敲除，

CRISPR/Cas9-mediated 目标集成在体内使用基于同源介导的端接连接策略

整合酶缺陷慢病毒载体载体的制备及其在细胞 CRISPR/Cas9-mediated 基因敲除中的表达

3 ' 快速扩增 CDNA 末端, 以图记录癌症

CRISPR 的染色质环结构重组

慢病毒载体介导的转基因小鼠生产: 一种简单高效的创始人直接研究方法

植物生长与农杆菌介导的植物浸水转化

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）

真菌快速缺失生产

摘要

探索更多视频

此视频中的章节

机器人平台的高通量原生质体分离和转型

饲养并在隐藏甲壳虫双链RNA介导的基因敲除，

CRISPR/Cas9-mediated 目标集成在体内使用基于同源介导的端接连接策略

整合酶缺陷慢病毒载体载体的制备及其在细胞 CRISPR/Cas9-mediated 基因敲除中的表达

3 ' 快速扩增 CDNA 末端, 以图记录癌症

CRISPR 的染色质环结构重组

慢病毒载体介导的转基因小鼠生产: 一种简单高效的创始人直接研究方法

植物生长与农杆菌介导的植物浸水转化

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）

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）