Name: plant promoter analysis: identification and characterization of root nodule specific promoter in the common bean
Uploaded: 2017-12-23T12:30:00.000+00:00
Duration: 10 min 58 s
Description: Watch this Scientific Journal Video about Plant Promoter Analysis: Identification and Characterization of Root Nodule Specific Promoter in the Common Bean at JoVE.com

这项工作得到了部分支持, 总署将军 Asuntos 个人 Académico、DGAPA/PAPIIT-大学 (赠款号:IN219916 到 m L 和 IA205117A) 和理事会全国 Ciencia y Tecnològia (委员会赠款240614至马丁)。

1. 识别、分离和克隆寻常型的促进者
<ol>
	<li>确定感兴趣基因的启动子序列。有几个基因组数据库和分析工具可用于植物, 如 Phytozome, Ensembl 植物, NCBI,等豆类启动子P. 寻常(PvNIN;Phvul. 009G115800) 在本研究中使用了20。</li>
	<li>设计寡为网关或传统限制酶克隆取决于所使用的向量系统 (图 1A)。 注: 标准 (25-35 bp), 盐水底漆工作正常。反向寡核苷酸序列, 在启动子和基因之间的侧翼是可取的。</li>
	<li>使用启动子特异性寡集, 从新近分离的P. 寻常基因组 DNA 中放大 PvNIN 启动子区域 (或感兴趣基因的启动子区域)。使用高保真聚合酶与以下 PCR 条件:95 ° c 为5分钟;30周期 (95 ° c 为三十年代, 55 ° c 为三十年代, 72 ° c 为 1 min);和72° c 为 5 min。</li>
	<li>运行放大片段1% 琼脂糖凝胶在 7 x 10 cm 凝胶托盘上, 并洗相应的增 (700 bp,图 1B) 和克隆到 pENTR/d-拓扑向量根据制造商的说明。</li>
	<li>使用制造商的说明和性兄弟 (LB) 补充50毫克/升卡那霉素 (Kan50) 的µL 150 µL, 将反应转化为合格的大肠杆菌细胞。在37° c 的夜间生长镀膜细胞。</li>
	<li>选择3-6 殖民地和文化过夜, 在 LB 培养基含有 Kan50。采用选择方法分离质粒 DNA, 用 PCR 法对质粒进行 M13 寡。用于 pcr, 使用 100 ng 质粒, 0.3 pm 寡, 10X pcr 缓冲, 0.5 U Taq DNA 聚合酶, 和10毫米 dNTPs。</li>
	<li>使用以下 PCR 扩增条件;95° c 为 3 min;30周期 (95 ° c 为三十年代, 58 ° c 为三十年代, 72 ° c 为七十五年代);72° c 为7分钟, 在1% 琼脂糖凝胶上可视化 PCR 产物。确保正确的插入有一个波段在 1024 bp 和没有插入的向量将有一个 324 bp 波段 (图 1C)。</li>
	<li>根据制造商的说明, 在输入向量 (pENTR/D-拓扑-PvNIN) 和目标矢量 pBGWFS7.021之间执行 LR 反应。</li>
	<li>将2µL 的 LR 反应转化为合格的大肠杆菌细胞, 使用标准技术, 如制造商的说明和板150µL 的转化混合物在 LB 补充与100毫克/升霉 (Spe100)。在37° c 的夜间生长镀膜细胞。</li>
	<li>选择大约5殖民地和文化他们过夜在 LB 培养基含有 Spe100。用选择的方法分离质粒 DNA, 利用启动子特异寡对质粒进行 PCR 分析。</li>
	<li>在步骤1.3 中使用 PCR 条件。在1% 琼脂糖凝胶上可视化 PCR 产品, 以确认放大。增是 700 bp。</li>
	<li>序列阳性质粒 pBGWFS7.0/PvNIN:: GUS-GFP (图 1D) 与启动子特定的寡验证插入的真实性。</li>
	<li>将质粒转化为杆菌应变 K599 (但是, 可以使用a. 杆菌的任何其他菌株)。添加2µL 的质粒准备到50µL electrocompetent 细胞和 electroporate 在一个1毫米试管与以下设置: 1.8 伏, 25 µF, 和200Ω。</li>
	<li>回收500µL 的 SOC 介质中的电池, 在28° c 下为 2 h 板上的 LB-Spe100, 在28° c 下生长2天。</li>
	<li>执行步骤1.7 所述的菌落筛选, 并使用步骤1.3 中的 PCR 条件。使甘油的库存从积极的克隆和存储在-80 ° c。</li>
	<li>使用杆菌区域性将空的 pBGWFS7.0 向量作为负控制。</li>
</ol>
2. a. 杆菌转换普通 Bean 的毛状根
<ol>
	<li>用50毫升的96% 乙醇为1分钟消毒豆类 (Jamapa) 种子 (约 20), 丢弃乙醇;然后加入50毫升20% 漂白剂 (次氯酸钠) 为10分钟, 在层流罩中不断混合。去除漂白剂, 在多余的无菌水中洗涤3次。 注意: 其他的寻常型品种可用于毛状根的诱导。</li>
	<li>在 #38 的无菌滤纸上萌发灭菌种子; 迪尔沃斯 (B 和 #38;D) 解决方案22 (表 1) 在黑暗中在28° c 的2天。</li>
	<li>转换前一天准备以下内容:
	<ol>
		<li>条纹出150µL 的杆菌区域性, 它窝藏二进制向量 (在步骤1.13 和1.14 中准备) 在固态 LB Spe100 上, 在一夜之间生长在28° c。</li>
		<li>用 B 和 #38;D 溶液填充15毫升管, 并用双层铝箔代替螺钉盖。组装成一个22厘米的玻璃管包含10毫升的 B 和 #38;D 解决方案 (图 2C) 和高压釜。</li>
	</ol>
	</li>
	<li>在转换的当天, 在层流罩中组装以下无菌材料: 从步骤2.2 萌发的种子, 从台阶2.3.1 的培养基, 2.3.2 的试管, 无菌双蒸馏水 (10 毫升), 管和吸管提示, 无菌钳,和3毫升注射器</li>
	<li>使用弯曲的200µL 尖将杆菌细胞从板上刮到离心管和重1毫升的双无菌水中。同样, 分别准备矢量控制单元。
	<ol>
		<li>将细胞轻轻地混合到重。不要涡流。</li>
	</ol>
	</li>
	<li>使用无菌吸管尖端在2.3.2 的管子的铝箔上制造一个 3 mm 的孔。</li>
	<li>收集的细胞 (无论是启动子或矢量控制) 从步骤2.5 与注射器和轻微刺痛胚的发芽种子与针尖 (图 2G)。 注意: 确保针穿透 (4-6 次) 的血管组织, 并注入小 (〜5µL) 滴的细胞在不同位置的伤口周围的下胚轴。</li>
	<li>小心地将注入苗的根部插入包含 B 和 #38;D 溶液中的 15 mL 管的 3 mm 孔中。返回整个安装到22毫升玻璃管和密封他们保持湿度。</li>
	<li>孵育在28° c 保持的生长腔中的管, 16 h light:8 的暗光周期。 注: 孵化后3-4 天, 植株生长到22毫升玻璃管的边缘。</li></ol>在这个阶段, 取下瓶盖, 用膜在阀杆周围封住边缘, 如图 2I所示。
	<li>在5-7 天内观察伤口部位的愈伤组织, 并在转化后10-12 天内发现2厘米毛状根。 注意: 在塑料和玻璃管中经常保持 B 和 #38;D 解决方案是很重要的。</li>
	<li>2周后, 通过切割茎2厘米以下的毛状根, 将原根移植到含有无菌蛭石的盆中。将植物保持在生长室 (16 小时 light:8 28 ° c), 用 B 和 #38;D 溶液冲洗。</li>

3. 启动子分析: 菜豆根瘤菌结节的启动子表达
<ol>
	<li>为诱导根瘤, 接种根瘤菌 tropici或根瘤菌 etli (与普通豆类相容) 在100毫升培养基 (0.5 克蛋白胨, 0.3 克酵母提取物) 补充与700毫米 CaCl2和20毫克毫升1啶酸孵育在30° c 为 24 h 与摇晃在300转每分钟。 注: 在转基因根瘤菌的情况下, 可以使用任何特定的抗生素。</li>
	<li>将离心机中的细胞在室温下为3分钟, 在10毫升的 10 mM MgSO4中重。通过稀释与 10 mM MgSO4, 在 od600时将单元格外径调整为0.6。</li>
	<li>接种1-2 毫升的培养 (从步骤 3.2) 每株 (促进剂和矢量控制) 诱导结节生长的毛状根。用有限的氮气 (2 mM3) #38;D 溶液灌溉植物以促进结瘤。</li>
	<li>根据实验需要, 收集不同时间点的毛状根。收集用于研究3-5 天后接种 (dpi) 的感染线索的样本。 注: 原始结核和幼结节可分别在 6-9 dpi 和10-12 天的 dpi 处进行研究。最后, 成熟和功能性结节可在 14-21 dpi、senesced 结节和 #62; 28 dpi。</li>
	<li>处理报告基因活动的根/结节 (GUS 或 GFP)。根据杰斐逊23描述的协议分析 GUS 活动。荧光显微镜下观察 GFP。用蓝色氩离子激光 (488 nm) 激发 GFP 荧光, 并从510到 540 nm 收集发射荧光。</li>
</ol>

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(49), e2633 (2011).</li><li>Kumagai, H., Kouchi, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+silencing+by+expression+of+hairpin+RNA+in+Lotus+japonicus+roots+and+root+nodules.">Gene silencing by expression of hairpin RNA in Lotus japonicus roots and root nodules.</a> Mol Plant Microbe Interact. 16 (8), 663-668 (2003).</li><li>Okamoto, S., Yoro, E., Suzaki, T., Kawaguchi, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hairy+Root+Transformation+in+Lotus+japonicus.">Hairy Root Transformation in Lotus japonicus.</a> Bio-protocol. 3 (12), e795 (2013).</li><li>Jacobs, T. B., Martin, G. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-throughput+CRISPR+Vector+Construction+and+Characterization+of+DNA+Modifications+by+Generation+of+Tomato+Hairy+Roots.">High-throughput CRISPR Vector Construction and Characterization of DNA Modifications by Generation of Tomato Hairy Roots.</a> J. Vis. Exp. (110), e53843 (2016).</li><li>Estrada-Navarrete, G., 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=Agrobacterium+rhizogenes-transformation+of+the+Phaseolus+spp.:+a+tool+for+functional+genomics.">Agrobacterium rhizogenes-transformation of the Phaseolus spp.: a tool for functional genomics.</a> Mol Plant Microbe Interact. 19 (12), 1385-1393 (2006).</li></ol>

作者没有什么可透露的。

在基因的功能分析中, 基因表达模式的研究对理解体内的基因的时空调控起着至关重要的作用。研究基因表达模式的一个众所周知的方法是克隆感兴趣的基因启动子区, 上游到报告基因如荧光标记基因 (GFP, RFP,等) 或β-酶。在此, 我们选择了根结节共生 (RNS) 特异性基因的启动子区域, 以研究在 RNS 中的P. ) 中的时空表达模式。选定的启动子序列已被克隆到上游的 GFP:: GUS 记者使用网关的方法成为启动子表达载体。
我们描述了在毛状根系统中的共生特异启动的表达.毛状根系统已被广泛应用于基因的功能表征, 包括 rna 介导的基因敲掉、基因的异位本构表达、启动子表达以及顽拗性作物的蛋白质定位。该系统的主要优点是容易、重现性好、可靠性高, 同时不耗费劳力。在其他农作物豆类中, 如G. max24、 m 蒺藜25、 L. 日本对虾26、27、番茄28、等, 都已成功采用必要的修改。此外, 该工具可用于其他豆类和 non-legume 品种, 当优化适当的杆菌菌株, 接种方法, 和生长条件。
有许多关键参数。在选择启动子区域时, 应注意设计寡, 以放大必要的启动子元素, 从翻译开始的上游区域开始 sitein 蛋白质编码基因;启动器分析应使用可用的软件, 以收集有关转录因子绑定到监管区域的信息。在诱导毛状根时要注意: 在适当的阶段注入幼苗;注射过程中, 确保针头不经过胚;并保持高湿条件。此外, 在研究毛状根的启动子表达谱时, 由A. 杆菌产生的毛状根并不总是被转换, 因此在使用 GFP 或 GUS 记者之前确认根的转换性质很重要。执行启动子表达式研究。应选择合适的菌株根瘤菌, 并根据议定书的规定调整根瘤菌的浓度。在适当的阶段进行取样接种对于记录 RNS 不同阶段启动子的时空表达是至关重要的。
杆菌转换的多毛根生产是在P.) 中生成复合植物的最快和最有效的替代方法之一。这些复合植物不能产生转基因种子, 但可以在几天内产生转基因根。在本协议中, 与其他方法29不同, 闭合管保持恒定的高湿度以在10天的 dpi 内更快地促进毛状根。

促进剂是重要的分子生物学工具, 在理解基因表达的调节方面起着至关重要的作用。发起人是基因序列的翻译起始密码子的上游的 DNA 序列, 它们携带着基因的中央调控信息;因此, 正确的注释和描述是理解基因功能的关键。根据表达模式, 植物促进剂被归类为本构、组织特异性, 或发展阶段特定和诱导的1。组技术的进步, 计算机建模的改进, 以及不同植物种类的基因组序列数量的增加, 促进了启动子序列的 large-scale 预测2。
另一方面, 建立启动子评估工具和遗传转化技术是快速、高效和可重现性的关键。与其他模型植物不同的是, 主要由于菜豆sp 的顽固性质, 导致了普通豆科豆类 (寻常型) 基因的功能特性。瞬变系统是一种替代的快速基因功能表征研究3。豆科植物共生研究中, 豆科寄主与根瘤菌细菌之间的相互作用是最易于操作的模型系统, 可用于结核特异基因的功能分析和促进剂研究。到目前为止, 与这些共生相关的几个豆类促进剂已经被描绘了, viz.,苜蓿蒺藜PT44, SWEET115,莲花日本的独眼巨人, UBQ6, VAG17, 甘氨酸最大 PT58, Exo70J9, 不寻常RbohB10,11,12, TRE113, PI3K14, TOR15,等。顺式元素直接影响基因调控。转录因子 ENBP1A 绑定到Cis管理区域 (−692 bp) 的早期瘤VfENOD12, 这有助于表达一个记者基因在结核原的蚕豆16。将结核特异性启动子 leghemoglobin GLB3 与异源截断促进剂δ-p35S 和δ-pNOS 的Cis管理区域 (−161 to −48 bp) 替换为结核特异性和减少启动子活性的损失17.
以前的报告显示, 在根毛细胞中启动根瘤菌感染需要转录因子, 对L. 日本对虾18中的根瘤发生器官也至关重要。在本研究中, 我们描述了一个协议的识别, 分离, 克隆, 并表征结核特异性启动剂在共同豆毛根。为了达到这一目的, 我们选择了一个根瘤菌的共生特异的一个. ....。此外, 该协议还描述了使用 a. 杆菌诱导的毛状根快速而多样的遗传转化系统。该系统在转化后不到2周内产生毛状根。最后, 用 GUS 染色法对根瘤菌定植的根瘤的时空表达进行了评价。
这里描述的过程不仅对于研究结瘤和根的豆科植物的11是有用的, 而且对于在根19中的启动子表达模式的研究也是有益的。此外, 本协议易于在非实验室中使用。

<table><tbody><tr><td>&lt;strong&gt;Primers for qRT-PCR assay&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>pNIN Forward</td><td>CACC ATA GCT CCC CAA AAT GGT AT</td><td></td><td></td></tr><tr><td>pNIN Reverse</td><td>CAT CTT CCT TCC ACT AAC TAA C</td><td></td><td></td></tr><tr><td>M13 Forward</td><td>GTA AAA CGA CGG CCA G</td><td></td><td></td></tr><tr><td>M13 Reverse</td><td>CAG GAA ACA GCT ATG AC</td><td></td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;REAGENTS&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>pENTR/D-TOPO Cloning Kit</td><td>Invitrogen</td><td>K243520</td><td></td></tr><tr><td>Gateway LR Clonase II Enzyme Mix&amp;nbsp;</td><td>Invitrogen</td><td>11791100</td><td></td></tr><tr><td>pBGWFS7.0&amp;nbsp;</td><td>Plant systems biology</td><td>https://gateway.psb.ugent.be/vector/show/pBGWFS7/search/index/</td><td></td></tr><tr><td>Platinum Taq DNA Polymerase</td><td>ThermoFisher Scientific</td><td>10966018</td><td></td></tr><tr><td>DNeasy Plant Mini Kit</td><td>Qiagen</td><td>69104</td><td></td></tr><tr><td>PureLink Quick Gel Extraction Kit</td><td>ThermoFisher Scientific</td><td>K210012</td><td></td></tr><tr><td>Platinum Pfx DNA Polymerase</td><td>Invitrogen</td><td>11708013</td><td></td></tr><tr><td>Certified Molecular Biology Agarose</td><td>Bio-Rad</td><td>1613102</td><td></td></tr><tr><td>One Shot TOP10 Chemically Competent E. coli</td><td>Invitrogen</td><td>C404006</td><td></td></tr><tr><td>Nacl</td><td>Sigma-Aldrich</td><td>S7653</td><td></td></tr><tr><td>Tryptone</td><td>Sigma-Aldrich</td><td>T7293-250G</td><td></td></tr><tr><td>Yeast extract</td><td>Sigma-Aldrich</td><td>Y1625-250G</td><td></td></tr><tr><td>Bacteriological agar</td><td>Sigma-Aldrich</td><td>A5306-1KG</td><td></td></tr><tr><td>Kanamycin sulfate</td><td>Sigma-Aldrich</td><td>60615-25G</td><td></td></tr><tr><td>Spectinomycin sulfate</td><td>Sigma-Aldrich</td><td>PHR1441</td><td></td></tr><tr><td>Ethyl alcohol</td><td>Sigma-Aldrich</td><td>E7023</td><td></td></tr><tr><td>Bacteriological peptone</td><td>Sigma-Aldrich</td><td>P0556</td><td></td></tr><tr><td>Calcium chloride</td><td>Sigma-Aldrich</td><td>C1016</td><td></td></tr><tr><td>Nalidixic acid</td><td>Sigma-Aldrich</td><td>N8878</td><td></td></tr><tr><td>Magnesium sulfate</td><td>Sigma-Aldrich</td><td>M7506</td><td></td></tr><tr><td>Gel loading solution</td><td>Sigma-Aldrich</td><td>G7654</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;EQUIPMENT&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Thermocycler</td><td>Veriti Thermal Cycler</td><td>4375786</td><td></td></tr><tr><td>Centrifuge</td><td>Sigma</td><td>Sigma 1-14K</td><td></td></tr><tr><td>Gel documentation unit</td><td>Carestream</td><td>Gel Logic 212 PRO</td><td></td></tr><tr><td>MaxQ SHKE6000 6000 Shaking Incubator - 115VAC</td><td>Thermo scientific</td><td>EW-51708-70</td><td></td></tr><tr><td>Plant growth chamber</td><td>MRC</td><td>PGI-550RH</td><td></td></tr><tr><td>Horizantal laminarair flow cabinate</td><td>Lumistell</td><td>LH-120</td><td></td></tr><tr><td>Fluorescent microscope</td><td>Leica</td><td>DM4500 B</td><td></td></tr><tr><td>Petridish</td><td>sym laboratorios</td><td>90X15</td><td></td></tr><tr><td>Scalpel Blade</td><td>Fisher scientific</td><td>53223</td><td></td></tr><tr><td>Falcon 15mL Conical Centrifuge Tubes</td><td>Fisher scientific</td><td>14-959-53A</td><td></td></tr><tr><td>22 mL glass tubes</td><td>Thomas scientific</td><td>45048-16150</td><td></td></tr></tbody></table>

plant promoter analysis: identification and characterization of root nodule specific promoter in the common bean

基因编码序列的上游序列被称为启动子序列。研究促进剂的表达模式对于了解靶基因的基因调控和时空表达模式具有重要意义。另一方面, 建立启动子评估工具和遗传转化技术是快速、高效和可重现性的关键。在本研究中, 我们研究了在转基因毛状根中, 根瘤菌共生特异性结节启动子 (菜豆) 的时空表达模式。利用植物基因组数据库和分析工具, 我们在转录融合中发现、分离和克隆了P. 寻常的启动子酶 (gus) gus 增强:: GFP。此外, 该协议还描述了一种快速而多样的遗传转化系统, 使用 农杆菌杆菌诱导的毛状根。该系统在转化后10至12天内产生≥2厘米毛状根。接下来, 我们评估了在 post-inoculation 的周期性时间间隔内, 在根瘤菌中接种多毛根的时空表达。GUS 活动的结果表明, 在结瘤过程中, 该启动子是活跃的。同时, 本协议还演示了如何识别、分离、克隆和描述在菜豆多毛根中的植物启动子。此外, 本协议易于在非实验室中使用。

本研究的目的是评估结核特异性P的时空表达模式。为此, 选择了寡基因的翻译起始密码子的上游 700 bp 区域, 并在 图 1A中描述了一组设计。使用高保真的聚合酶, 该在一级启动子片段被放大, 孤立 (图 1B), 并通过网关方法克隆到目标二进制向量 pBGWFS7.0 (图 1C), 从而生成一个转录融合到嵌合记者 gus 增强 gfp (pBGWFS7.0/PvNIN:: gus-gfp)。pBGWFS7.0/PvNIN:: GUS-GFP 矢量结构通常生成数以百计的独立克隆。PCR 与基因特异寡的菌落筛选容易识别正确的克隆 (图 2D)。PCR 阳性质粒与启动子特异寡测序, 以验证插入物的真实性。
在整个实验中保持高湿度是诱导愈伤组织和毛状根的必要条件。在受伤部位, 愈伤组织通常在5至7天 (图 2J), 在转化后10至12天观察到2厘米毛状根。大多数A. 杆菌将植物成功地转化为2周 (图 2K) 产生毛状根。然而, 毛状根的数量和长度可以是可变的。因此, 选择最有力的生长根进行分析和进一步的实验。通过在毛根部下面切割2厘米的茎, 将其移植到含有无菌蛭石 (图 2L) 或水培系统中的花盆中。
为了研究结核特异性启动子的时空表达, 用R. tropici接种了多毛根, 根据实验需要, post-inoculation 周期性观察 GUS 活动。接种与R. tropici诱导在转基因根强 GUS 表达, 表明根瘤菌诱导在共同 bean (图 3A) 中的表达。此外, 6-9 dpi 的取样显示了根瘤菌感染的根毛细胞 (图 3B) 中的 GUS 表达式。菜豆结核原, 幼结节和成熟的结核也显示了表达 (图 3C)。在结瘤的所有阶段, 在矢量控制根 (图 3D, 3E, 3F) 中都没有看到这种 GUS 表达式。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56140/56140fig1.jpg"> 
图 1:大纲P.. 基因结构和克隆.(a)万年基因结构的示意图表示, 显示 5 "UTR (绿色)、4显、3内含子和 3" UTR (红色) 在P 寻常的染色体编号9上。在 ATG 转换起始密码子的上游, 显示了寡设计的启动区域。FO, 向前寡聚;反渗透, 反向寡聚;ATG, 启动密码子;TAA, 停止密码。(B) PvNIN 启动子区域是由新的孤立的P. 寻常基因组 DNA 使用启动子特异性寡集放大的。M, 1 Kb 梯子;1、PCR 反应与 gDNA 显示增 700 bp 的大小;2、PCR 反应无 gDNA (ve 控制)。(C) 通过 PCR 方法对 pENTR/d-拓扑 PvNIN 质粒进行筛选, 采用矢量特异 M13 寡。正确插入有一个波段在 1024 bp (车道 3-6) 和载体没有插入将有一个 324 bp 带 (车道 1-2)。(D) 在本研究中使用的启动子表达式二进制矢量图, pBGWFS7.0 向量显示 PvNIN 启动子与 GUS 和 GFP 的融合。<a href="//ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/56140/56140fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56140/56140fig2.jpg"> 
图 2:杆菌转换了普通 bean 的毛状根.(A) 在无菌滤纸上发芽的黑 Jamapa 的种子. (B) 种子, 脱去外衣。(C) 管道设置为毛状根诱导。(D) 刮出杆菌区域性 (普宁和 #38; 控件)。(E)杆菌区域性悬浮在无菌蒸馏水中。(F, G)将细菌细胞注入 hypocotyles。(H) 管道设置与菜豆苗注入细菌细胞。(I, J)受伤部位 5-7 dpi 形成的愈伤组织。(K) 毛状根 15 dpi (L) 将水龙头根部2厘米以下的毛状根诱导点切除。(M) 将移植到无菌蛭石中的复合植株与R. tropici一起接种。<a href="//ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/56140/56140fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56140/56140fig3.jpg"> 
图 3: 启动子项分析p. )在转基因 p. 寻常根.promoterNIN 显示的空间和时间模式 "表达式:: gus-GFP 在以 gus 为基质的转基因毛状根中的构建. PvNIN的光学显微镜图像: gus: (A) 根接种与R. tropici, (B) gus 表达在卷曲的根毛, 和 (C) 年轻的结节。控件的图像 (空向量) 在 (D) 中不显示此类 GUS 表达式 (以R. tropici、(E) 卷曲的根毛和 (F) 幼结节接种。Rh 根头发<a href="//ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/56140/56140fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

Watch this Scientific Journal Video about Plant Promoter Analysis: Identification and Characterization of Root Nodule Specific Promoter in the Common Bean at JoVE.com

Plant Promoter Analysis: Identification and Characterization of Root Nodule Specific Promoter in the Common Bean

植物启动子分析: 菜豆根瘤特异启动子的鉴定与表征

启动子表达分析对于提高对基因调控的理解和靶基因的时空表达至关重要。在这里, 我们提出了一个协议, 以确定, 隔离和克隆植物启动子。此外, 我们还描述了在菜豆多毛根中的根瘤特异启动子的特性。

The upstream sequences of gene coding sequences are termed as promoter sequences. Studying the expression patterns of promoters are very significant in ...

plant-promoter-analysis-identification-characterization-root-nodule

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11.7K 次观看.  Escuela Nacional de Estudios Superiores Unidad León- Universidad Nacional Autónoma de México (UNAM). 启动子表达分析对于提高对基因调控的理解和靶基因的时空表达至关重要。在这里, 我们提出了一个协议, 以确定, 隔离和克隆植物启动子。此外, 我们还描述了在菜豆多毛根中的根瘤特异启动子的特性。

视频: Plant Promoter Analysis: Identification and Characterization of Root Nodule Specific Promoter in the Common Bean

An Optimized Rhizobox Protocol to Visualize Root Growth and Responsiveness to Localized Nutrients

Roots are notoriously difficult to study. Soil is both a visual and mechanical barrier, making it difficult to track roots in situ without destructive harvest or expensive equipment. We present a customizable and affordable rhizobox method that allows the non-destructive visualization of root growth over time and is particularly well-suited to studying root plasticity in response to localized resource patches. The method was validated by assessing maize genotypic variation in plasticity responses to patches containing 15N-labeled legume residue. Methods are described to obtain representative developmental measurements over time, measure root length density in resource-containing and control patches, calculate root growth rates, and determine 15N recovery by plant roots and shoots. Advantages, caveats, and potential future applications of the method are also discussed. Although care must be taken to ensure that experimental conditions do not bias root growth data, the rhizobox protocol presented here yields reliable results if carried out with sufficient attention to detail.

Visualizing and measuring root growth in situ is extremely challenging. We present a customizable rhizobox method to track root development and proliferation over time in response to nutrient enrichment. This method is used to analyze maize genotypic differences in root plasticity in response to an organic nitrogen source.

一种优化的 Rhizobox 协议, 用于可视化根系生长和对局部营养素的响应

Roots are notoriously difficult to study. Soil is both a visual and mechanical barrier, making it difficult to track roots in situ without destructive ...

科研

环境科学

Agrobacterium tumefaciens and Agrobacterium rhizogenes-Mediated Transformation of Potato and the Promoter Activity of a Suberin Gene by GUS Staining

Agrobacterium sp. is one of the most widely used methods to obtain transgenic plants as it has the ability to transfer and integrate its own T-DNA into the plant&#39;s genome. Here, we present two transformation systems to genetically modify potato (Solanum tuberosum) plants. In A. tumefaciens transformation, leaves are infected, the transformed cells are selected and a new complete transformed plant is regenerated using phytohormones in 18 weeks. In A. rhizogenes transformation, stems are infected by injecting the bacteria with a needle, the new emerged transformed hairy roots are detected using a red fluorescent marker and the non-transformed roots are removed. In 5-6 weeks, the resulting plant is a composite of a wild type shoot with fully developed transformed hairy roots. To increase the biomass, the transformed hairy roots can be excised and self-propagated. We applied both Agrobacterium-mediated transformation methods to obtain roots expressing the GUS reporter gene driven by a suberin biosynthetic gene promoter. The GUS staining procedure is provided and allows the cell localization of the promoter induction. In both methods, the transformed potato roots showed GUS staining in the suberized endodermis and exodermis, and additionally, in A. rhizogenes transformed roots the GUS activity was also detected in the emergence of lateral roots. These results suggest that A. rhizogenes can be a fast alternative tool to study the genes that are expressed&#160;in roots.

Agrobacterium tumefaciens and Agrobacterium rhizogenes-Mediated Transformation of Potato and the Promoter Activity of a Suberin Gene by GUS Staining

Here, we present two protocols to transform potato plants. The Agrobacterium tumefaciens transformation leads to a complete transgenic plant while the Agrobacterium rhizogenes produces transgenic hairy roots in a wild type shoot that can be self-propagated. We then detect promoter activity by GUS staining in the transformed roots.

农杆菌和根瘤菌--马铃薯介导的马铃薯转化与葡萄染色诱导素基因的启动子活性

Agrobacterium sp. is one of the most widely used methods to obtain transgenic plants as it has the ability to transfer and integrate its own T-DNA into ...

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 ...

遗传学

Translating Ribosome Affinity Purification (TRAP) to Investigate Arabidopsis thaliana Root Development at a Cell Type-Specific Scale

In this article, we give hands-on instructions to obtain translatome data from different Arabidopsis thaliana root cell types via the translating ribosome affinity purification (TRAP) method and consecutive optimized low-input library preparation.
As starting material, we employ plant lines that express GFP-tagged ribosomal protein RPL18 in a cell type-specific manner by use of adequate promoters. Prior to immunopurification and RNA extraction, the tissue is snap frozen, which preserves tissue integrity and simultaneously allows execution of time series studies with high temporal resolution. Notably, cell wall structures remain intact, which is a major drawback in alternative procedures such as fluorescence-activated cell sorting-based approaches that rely on tissue protoplasting to isolate distinct cell populations. Additionally, no tissue fixation is necessary as in laser capture microdissection-based techniques, which allows high-quality RNA to be obtained.
However, sampling from subpopulations of cells and only isolating polysome-associated RNA severely limits RNA yields. It is, therefore, necessary to apply sufficiently sensitive library preparation methods for successful data acquisition by RNA-seq.
TRAP offers an ideal tool for plant research as many developmental processes involve cell wall-related and mechanical signaling pathways. The use of promoters to target specific cell populations is bridging the gap between organ and single-cell level that in turn suffer from little resolution or very high costs. Here, we apply TRAP to study cell-cell communication in lateral root formation.

Translating Ribosome Affinity Purification TRAP to Investigate Arabidopsis thaliana Root Development at a Cell Type-Specific Scale

Translating ribosome affinity purification (TRAP) offers the possibility to dissect developmental programs with minimal processing of organs and tissues. The protocol yields high-quality RNA from cells targeted with a green fluorescent protein (GFP)-labeled ribosomal subunit. Downstream analysis tools, such as qRT-PCR or RNA-seq, reveal tissue and cell type-specific expression profiles.

翻译核糖体亲和纯化（TRAP）以研究细胞类型特异性尺度下阿拉伯生物基塔利亚纳根发育

In this article, we give hands-on instructions to obtain translatome data from different Arabidopsis thaliana root cell types via the translating ribosome ...

发育生物学

Polysome Purification from Soybean Symbiotic Nodules

The aim of this protocol is to provide a strategy for studying the eukaryotic translatome of the soybean (Glycine max) symbiotic nodule. This paper describes methods optimized to isolate plant-derived polyribosomes and their associated mRNAs to be analyzed using RNA-sequencing. First, cytoplasmic lysates are obtained through homogenization in polysome- and RNA-preserving conditions from whole, frozen soybean nodules. Then, lysates are cleared by low-speed centrifugation, and 15% of the supernatant is used for total RNA (TOTAL) isolation. The remaining cleared lysate is used to isolate polysomes by ultracentrifugation through a two-layer sucrose cushion (12% and 33.5%). Polysome-associated mRNA (PAR) is purified from polysomal pellets after resuspension. Both TOTAL and PAR are evaluated by highly sensitive capillary electrophoresis to meet the quality standards of sequencing libraries for RNA-seq. As an example of a downstream application, after sequencing, standard pipelines for gene expression analysis can be used to obtain differentially expressed genes at the transcriptome and translatome levels. In summary, this method, in combination with RNA-seq, allows the study of the translational regulation of eukaryotic mRNAs in a complex tissue such as the symbiotic nodule.

This protocol describes a method for eukaryotic polysome purification from intact soybean nodules. After sequencing, standard pipelines for gene expression analysis can be used to identify differentially expressed genes at the transcriptome and translatome levels.

大豆共生结节多聚体纯化

The aim of this protocol is to provide a strategy for studying the eukaryotic translatome of the soybean (Glycine max) symbiotic nodule. This paper ...

Generation of Composite Plants in Medicago truncatula used for Nodulation Assays

Similar to Agrobacterium tumerfaciens, Agrobacterium rhizogenes can transfer foreign DNAs into plant cells based on the autonomous root-inducing (Ri) plasmid. A. rhizogenes can cause hairy root formation on plant tissues and form composite plants after transformation. On these composite plants, some of the regenerated roots are transgenic, carrying the wild type T-DNA and the engineered binary vector; while the shoots are still non-transgenic, serving to provide energy and growth support. These hairy root composite plants will not produce transgenic seeds, but there are a number of important features that make these composite plants very useful in plant research. First, with a broad host range,A. rhizogenes can transform many plant species, especially dicots, allowing genetic engineering in a variety of species. Second, A. rhizogenes infect tissues and explants directly; no tissue cultures prior to transformation is necessary to obtain composite plants, making them ideal for transforming recalcitrant plant species. Moreover, transgenic root tissues can be generated in a matter of weeks. For Medicago truncatula, we can obtain transgenic roots in as short as three weeks, faster than normal floral dip Arabidopsis transformation. Overall, the hairy root composite plant technology is a versatile and useful tool to study gene functions and root related-phenotypes. Here we demonstrate how hairy root composite plants can be used to study plant-rhizobium interactions and nodulation in the difficult-to-transform species M. truncatula.

Generation of Composite Plants in Medicago truncatula used for Nodulation Assays

We demonstrate how hairy root composite plants can be used to study plant-rhizobium interactions and nodulation in the difficult-to-transform species Medicago truncatula.

复合植物代苜蓿truncatula</em用于检测结瘤

Similar to Agrobacterium tumerfaciens, Agrobacterium rhizogenes can transfer foreign DNAs into plant cells based on the autonomous root-inducing (Ri) ...

生物学

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 ...

生物工程

Lateral Root Inducible System in Arabidopsis and Maize

Lateral root development contributes significantly to the root system, and hence is crucial for plant growth. The study of lateral root initiation is however tedious, because it occurs only in a few cells inside the root and in an unpredictable manner. To circumvent this problem, a Lateral Root Inducible System (LRIS) has been developed. By treating seedlings consecutively with an auxin transport inhibitor and a synthetic auxin, highly controlled lateral root initiation occurs synchronously in the primary root, allowing abundant sampling of a desired developmental stage. The LRIS has first been developed for Arabidopsis thaliana, but can be applied to other plants as well. Accordingly, it has been adapted for use in maize (Zea mays). A detailed overview of the different steps of the LRIS in both plants is given. The combination of this system with comparative transcriptomics made it possible to identify functional homologs of Arabidopsis lateral root initiation genes in other species as illustrated here for the CYCLIN B1;1 (CYCB1;1) cell cycle gene in maize. Finally, the principles that need to be taken into account when an LRIS is developed for other plant species are discussed.

Lateral Root Inducible System in Arabidopsis and Maize

The Lateral Root Inducible System (LRIS) allows for synchronous induction of lateral roots and is presented for Arabidopsis thaliana and maize.

侧根诱导系统<em&gt;拟南芥</em&gt;和玉米

Lateral root development contributes significantly to the root system, and hence is crucial for plant growth. The study of lateral root initiation is ...

植物启动子分析: 菜豆根瘤特异启动子的鉴定与表征

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此视频中的章节

一种优化的 Rhizobox 协议, 用于可视化根系生长和对局部营养素的响应

农杆菌和根瘤菌--马铃薯介导的马铃薯转化与葡萄染色诱导素基因的启动子活性

CcCIPK14 Gene Function Analysis to Illuminate the Efficient Root Transgenic System

翻译核糖体亲和纯化（TRAP）以研究细胞类型特异性尺度下阿拉伯生物基塔利亚纳根发育

大豆共生结节多聚体纯化

复合植物代苜蓿truncatula

大豆毛根转化基因功能分析

大豆毛根转化基因功能分析

大豆毛根转化基因功能分析

侧根诱导系统