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 border="0" cellpadding="0" cellspacing="0" width="1125">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">Primers for qRT-PCR assay</td>
			<td style="width:253px;"></td>
			<td style="width:460px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">pNIN Forward</td>
			<td>CACC ATA GCT CCC CAA AAT GGT AT</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:245px;">pNIN Reverse</td>
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			<td height="21" style="height:21px;width:245px;">M13 Forward</td>
			<td>GTA AAA CGA CGG CCA G</td>
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			<td height="21" style="height:21px;width:245px;">M13 Reverse</td>
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			<td height="22" style="height:22px;">pBGWFS7.0&#160;</td>
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			<td height="21" style="height:21px;width:245px;">DNeasy Plant Mini Kit</td>
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			<td height="21" style="height:21px;width:245px;">Centrifuge</td>
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			<td height="21" style="height:21px;width:245px;">Gel documentation unit</td>
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			<td height="42" style="height:42px;width:245px;">MaxQ SHKE6000 6000 Shaking Incubator - 115VAC</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:245px;">Plant growth chamber</td>
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			<td height="21" style="height:21px;width:245px;">Horizantal laminarair flow cabinate</td>
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			<td height="21" style="height:21px;width:245px;">Fluorescent microscope</td>
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			<td style="width:460px;">DM4500 B</td>
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			<td height="21" style="height:21px;width:245px;">Petridish</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:245px;">Scalpel Blade&#160;</td>
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			<td height="42" style="height:42px;width:245px;">Falcon 15mL Conical Centrifuge Tubes</td>
			<td style="width:253px;">Fisher scientific</td>
			<td style="width:460px;">14-959-53A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">22 mL glass tubes</td>
			<td style="width:253px;">Thomas scientific</td>
			<td style="width:460px;">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.remotexs.ntu.edu.sg/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.remotexs.ntu.edu.sg/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.remotexs.ntu.edu.sg/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

Nanyang Technological University

Research

JoVE Journal

Biology

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

Identification and Characterization of Protein Glycosylation using Specific Endo- and Exoglycosidases

Glycosylation, the addition of covalently linked sugars, is a major post-translational modification of proteins that can significantly affect processes such as cell adhesion, molecular trafficking, clearance, and signal transduction1-4. In eukaryotes, the most common glycosylation modifications in the secretory pathway are additions at consensus asparagine residues (N-linked); or at serine or threonine residues (O-linked) (Figure 1). Initiation of N-glycan synthesis is highly conserved in eukaryotes, while the end products can vary greatly among different species, tissues, or proteins. Some glycans remain unmodified ("high mannose N-glycans") or are further processed in the Golgi ("complex N-glycans"). Greater diversity is found for O-glycans, which start with a common N-Acetylgalactosamine (GalNAc) residue in animal cells but differ in lower organisms1.
The detailed analysis of the glycosylation of proteins is a field unto itself and requires extensive resources and expertise to execute properly. However a variety of available enzymes that remove sugars (glycosidases) makes possible to have a general idea of the glycosylation status of a protein in a standard laboratory setting. Here we illustrate the use of glycosidases for the analysis of a model glycoprotein: recombinant human chorionic gonadotropin beta (hCG&beta;), which carries two N-glycans and four O-glycans 5. The technique requires only simple instrumentation and typical consumables, and it can be readily adapted to the analysis of multiple glycoprotein samples.
Several enzymes can be used in parallel to study a glycoprotein. PNGase F is able to remove almost all types of N-linked glycans6,7. For O-glycans, there is no available enzyme that can cleave an intact oligosaccharide from the protein backbone. Instead, O-glycans are trimmed by exoglycosidases to a short core, which is then easily removed by O-Glycosidase. The Protein Deglycosylation Mix contains PNGase F, O-Glycosidase, Neuraminidase (sialidase), &beta;1-4 Galactosidase, and &beta;-N-Acetylglucosaminidase. It is used to simultaneously remove N-glycans and some O-glycans8 . Finally, the Deglycosylation Mix was supplemented with a mixture of other exoglycosidases (&alpha;-N-Acetylgalactosaminidase, &alpha;1-2 Fucosidase, &alpha;1-3,6 Galactosidase, and &beta;1-3 Galactosidase ), which help remove otherwise resistant monosaccharides that could be present in certain O-glycans.
SDS-PAGE/Coomasie blue is used to visualize differences in protein migration before and after glycosidase treatment. In addition, a sugar-specific staining method, ProQ Emerald-300, shows diminished signal as glycans are successively removed. This protocol is designed for the analysis of small amounts of glycoprotein (0.5 to 2 &mu;g), although enzymatic deglycosylation can be scaled up to accommodate larger quantities of protein as needed.

Using specific glycosidases to remove sugars from glycoproteins followed by SDS-PAGE is a valuable method to detect glycan modifications on protein samples and is a good choice for initial glycobiology studies. Changes following deglycosylation can be detected as shifts in gel mobility or by staining with glycan sensitive reagents.

使用具体远藤和exoglycosidases糖基化蛋白质的鉴定和表征

Glycosylation, the addition of covalently linked sugars, is a major post-translational modification of proteins that can significantly affect processes ...

科研

生物学

Fluorescence-microscopy Screening and Next-generation Sequencing: Useful Tools for the Identification of Genes Involved in Organelle Integrity

This protocol describes a fluorescence microscope-based screening of Arabidopsis seedlings and describes how to map recessive mutations that alter the subcellular distribution of a specific tagged fluorescent marker in the secretory pathway. Arabidopsis is a powerful biological model for genetic studies because of its genome size, generation time, and conservation of molecular mechanisms among kingdoms. The array genotyping as an approach to map the mutation in alternative to the traditional method based on molecular markers is advantageous because it is relatively faster and may allow the mapping of several mutants in a really short time frame. This method allows the identification of proteins that can influence the integrity of any organelle in plants. Here, as an example, we propose a screen to map genes important for the integrity of the endoplasmic reticulum (ER). Our approach, however, can be easily extended to other plant cell organelles (for example see1,2), and thus represents an important step toward understanding the molecular basis governing other subcellular structures.

A fundamental quest in cell biology is to define the mechanisms that underlie the identity of the organelles that make eukaryotic cells. Here we propose a method to identify the genes responsible for the morphological and functional integrity of plant organelles using fluorescence microscopy and next-generation sequencing tools.

荧光显微镜筛选和新一代测序:参与细胞器完整性的基因鉴定的有用工具

This protocol describes a fluorescence microscope-based screening of Arabidopsis seedlings and describes how to map recessive mutations that alter the ...

Pyrosequencing for Microbial Identification and Characterization

Pyrosequencing is a versatile technique that facilitates microbial genome sequencing that can be used to identify bacterial species, discriminate bacterial strains and detect genetic mutations that confer resistance to anti-microbial agents. The advantages of pyrosequencing for microbiology applications include rapid and reliable high-throughput screening and accurate identification of microbes and microbial genome mutations. Pyrosequencing involves sequencing of DNA by synthesizing the complementary strand a single base at a time, while determining the specific nucleotide being incorporated during the synthesis reaction. The reaction occurs on immobilized single stranded template DNA where the four deoxyribonucleotides (dNTP) are added sequentially and the unincorporated dNTPs are enzymatically degraded before addition of the next dNTP to the synthesis reaction. Detection of the specific base incorporated into the template is monitored by generation of chemiluminescent signals. The order of dNTPs that produce the chemiluminescent signals determines the DNA sequence of the template. The real-time sequencing capability of pyrosequencing technology enables rapid microbial identification in a single assay. In addition, the pyrosequencing instrument, can analyze the full genetic diversity of anti-microbial drug resistance, including typing of SNPs, point mutations, insertions, and deletions, as well as quantification of multiple gene copies that may occur in some anti-microbial resistance patterns.

Pyrosequencing is a versatile technique that facilitates microbial genome sequencing that can be used to identify bacterial species, discriminate bacterial strains, and detect genetic mutations that confer resistance to anti-microbial agents. In this video, the procedure for microbial amplicon generation, amplicon pyrosequencing, and DNA sequence analysis will be demonstrated.

焦磷酸测序的微生物鉴定和表征

Pyrosequencing is a versatile technique that facilitates microbial genome sequencing that can be used to identify bacterial species, discriminate ...

Identification of Post-translational Modifications of Plant Protein Complexes

Plants adapt quickly to changing environments due to elaborate perception and signaling systems. During pathogen attack, plants rapidly respond to infection via the recruitment and activation of immune complexes. Activation of immune complexes is associated with post-translational modifications (PTMs) of proteins, such as phosphorylation, glycosylation, or ubiquitination. Understanding how these PTMs are choreographed will lead to a better understanding of how resistance is achieved.
Here we describe a protein purification method for nucleotide-binding leucine-rich repeat (NB-LRR)-interacting proteins and the subsequent identification of their post-translational modifications (PTMs). With small modifications, the protocol can be applied for the purification of other plant protein complexes. The method is based on the expression of an epitope-tagged version of the protein of interest, which is subsequently partially purified by immunoprecipitation and subjected to mass spectrometry for identification of interacting proteins and PTMs.
This protocol demonstrates that: i). Dynamic changes in PTMs such as phosphorylation can be detected by mass spectrometry; ii). It is important to have sufficient quantities of the protein of interest, and this can compensate for the lack of purity of the immunoprecipitate; iii). In order to detect PTMs of a protein of interest, this protein has to be immunoprecipitated to get a sufficient quantity of protein.

We describe here a protocol for the purification and characterization of plant protein complexes. We demonstrate that by immunoprecipitating a single protein within a complex, so we can identify its post-translational modifications and its interacting partners.

植物蛋白复合体的翻译后修饰的鉴定

Plants adapt quickly to changing environments due to elaborate perception and signaling systems. During pathogen attack, plants rapidly respond to ...

Generation of Plasmid Vectors Expressing FLAG-tagged Proteins Under the Regulation of Human Elongation Factor-1&#945; Promoter Using Gibson Assembly

Gibson assembly (GA) cloning offers a rapid, reliable, and flexible alternative to conventional DNA cloning methods. We used GA to create customized plasmids for expression of exogenous genes in mouse embryonic stem cells (mESCs). Expression of exogenous genes under the control of the SV40 or human cytomegalovirus promoters diminishes quickly after transfection into mESCs. A remedy for this diminished expression is to use the human elongation factor-1 alpha (hEF1&#945;) promoter to drive gene expression. Plasmid vectors containing hEF1&#945; are not as widely available as SV40- or CMV-containing plasmids, especially those also containing N-terminal 3xFLAG-tags. The protocol described here is a rapid method to create plasmids expressing FLAG-tagged CstF-64 and CstF-64 mutant under the expressional regulation of the hEF1&#945; promoter. GA uses a blend of DNA exonuclease, DNA polymerase and DNA ligase to make cloning of overlapping ends of DNA fragments possible. Based on the template DNAs we had available, we designed our constructs to be assembled into a single sequence. Our design used four DNA fragments: pcDNA 3.1 vector backbone, hEF1&#945; promoter part 1, hEF1&#945; promoter part 2 (which contained 3xFLAG-tag purchased as a double-stranded synthetic DNA fragment), and either CstF-64 or specific CstF-64 mutant. The sequences of these fragments were uploaded to a primer generation tool to design appropriate PCR primers for generating the DNA fragments. After PCR, DNA fragments were mixed with the vector containing the selective marker and the GA cloning reaction was assembled. Plasmids from individual transformed bacterial colonies were isolated. Initial screen of the plasmids was done by restriction digestion, followed by sequencing. In conclusion, GA allowed us to create customized plasmids for gene expression in 5 days, including construct screens and verification.

Synthesis of custom plasmids is labor and time consuming. This protocol describes the use of Gibson assembly cloning to reduce the work and duration of custom DNA cloning procedure. The protocol described also produces reliable tagged protein constructs for mammalian expression at similar cost to the traditional cut-and-paste DNA cloning.

质粒载体产生表达FLAG标记的蛋白质在人延伸因子1α启动子的使用吉布森大会规则

Gibson assembly (GA) cloning offers a rapid, reliable, and flexible alternative to conventional DNA cloning methods. We used GA to create customized ...

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 ...

Methods to Discover Alternative Promoter Usage and Transcriptional Regulation of Murine Bcrp1

Gene expression in different tissues is often controlled by alternative promoters that result in the synthesis of mRNA with unique &#8212; usually untranslated &#8212; first exons. Bcrp1 (Abcg2), the murine orthologue of the ABC transporter Breast Cancer Resistance Protein (BCRP, ABCG2), has at least four alternative promoters that are designated by the corresponding four alternative first exons produced: E1U, E1A, E1B, and E1C. Herein, in-silico protocols are presented to predict alternative promoter usage for Bcrp1. Furthermore, reporter assay methods are described to produce reporter constructs for alternative promoters and to determine the functionality of putative promoters upstream of the alternative first exons that are identified.

With the murine ABC transporter Bcrp1 (Abcg2) as an example, in-silico protocols are presented to detect alternative promoter usage in genes expressed in mouse tissues, and to evaluate the functionality of the alternative promoters identified using reporter assays.

方法发现小鼠BCRP1的另类用法子和转录调控

Gene expression in different tissues is often controlled by alternative promoters that result in the synthesis of mRNA with unique &#8212; usually ...

Identification of Novel Regulators of Plant Transpiration by Large-Scale Thermal Imaging Screening in Helianthus Annuus

Plant adaptation to biotic and abiotic stresses is governed by a variety of factors, among which the regulation of stomatal aperture in response to water deficit or pathogens plays a crucial role. Identifying small molecules that regulate stomatal movement can therefore contribute to understanding the physiological basis by which plants adapt to their environment. Large-scale screening approaches that have been used to identify regulators of stomatal movement have potential limitations: some rely heavily on the abscisic acid (ABA) hormone signaling pathway, therefore excluding ABA-independent mechanisms, while others rely on the observation of indirect, long-term physiological effects such as plant growth and development. The screening method presented here allows the large-scale treatment of plants with a library of chemicals coupled with a direct quantification of their transpiration by thermal imaging. Since evaporation of water through transpiration results in leaf surface cooling, thermal imaging provides a non-invasive approach to investigate changes in stomatal conductance over time. In this protocol, Helianthus annuus seedlings are grown hydroponically and then treated by root feeding, in which the primary root is cut and dipped into the chemical being tested. Thermal imaging followed by statistical analysis of cotyledonary temperature changes over time allows for the identification of bioactive molecules modulating stomatal aperture. Our proof-of-concept experiments demonstrate that a chemical can be carried from the cut root to the cotyledon of the sunflower seedling within 10 minutes. In addition, when plants are treated with ABA as a positive control, an increase in leaf surface temperature can be detected within minutes. Our method thus allows the efficient and rapid identification of novel molecules regulating stomatal aperture.

Identification of Novel Regulators of Plant Transpiration by Large-Scale Thermal Imaging Screening in Helianthus Annuus

We provide a method for identifying modulators of foliar transpiration by large-scale screening of a compound library.

赫连图斯安努斯大型热成像筛选装置新稳压器的鉴定

Plant adaptation to biotic and abiotic stresses is governed by a variety of factors, among which the regulation of stomatal aperture in response to water ...

Laser-Capture Microdissection RNA-Sequencing for Spatial and Temporal Tissue-Specific Gene Expression Analysis in Plants

The development of a complex multicellular organism is governed by distinct cell types that have different transcriptional profiles. To identify transcriptional regulatory networks that govern developmental processes it is necessary to measure the spatial and temporal gene expression profiles of these individual cell types. Therefore, insight into the spatio-temporal control of gene expression is essential to gain understanding of how biological and developmental processes are regulated. Here, we describe a laser-capture microdissection (LCM) method to isolate small number of cells from three barley embryo organs over a time-course during germination followed by transcript profiling. The method consists of tissue fixation, tissue processing, paraffin embedding, sectioning, LCM and RNA extraction followed by real-time PCR or RNA-seq. This method has enabled us to obtain spatial and temporal profiles of seed organ transcriptomes from varying numbers of cells (tens to hundreds), providing much greater tissue-specificity than typical bulk-tissue analyses. From these data we were able to define and compare transcriptional regulatory networks as well as predict candidate regulatory transcription factors for individual tissues. The method should be applicable to other plant tissues with minimal optimization.

Presented here is a protocol for laser-capture microdissection (LCM) of plant tissues. LCM is a microscopic technique for isolating areas of tissue in a contamination-free manner. The procedure includes tissue fixation, paraffin embedding, sectioning, LCM and RNA extraction. RNA is used in the downstream tissue-specific, temporally resolved analysis of transcriptomes.

激光捕获微分块RNA-测序空间和时间组织特异性基因表达分析

The development of a complex multicellular organism is governed by distinct cell types that have different transcriptional profiles. To identify ...

Divergence of Root Microbiota in Different Habitats based on Weighted Correlation Networks

The root microbiome plays an important role in plant growth and environmental adaptation. Network analysis is an important tool for studying communities, which can effectively explore the interaction relationship or co-occurrence model of different microbial species in different environments. The purpose of this manuscript is to provide details on how to use the weighted correlation network algorithm to analyze different co-occurrence networks that may occur in microbial communities due to different ecological environments. All analysis of the experiment is performed in the WGCNA package. WGCNA is an R package for weighted correlation network analysis. The experimental data used to demonstrate these methods were microbial community data from the NCBI (National Center for Biotechnology Information) database for three niches of the rice (Oryza sativa) root system. We used the weighted correlation network algorithm to construct co-abundance networks of microbial community in each of the three niches. Then, differential co-abundance networks among endosphere, rhizoplane and rhizosphere soil were identified. In addition, the core genera in network were obtained by the "WGCNA" package, which plays an important regulated role in network functions. These methods enable researchers to analyze the response of microbial network to environmental disturbance and verify different microbial ecological response theories. The results of these methods show that the significant differential microbial networks identified in the endosphere, rhizoplane and rhizosphere soil of rice.

Network analysis was applied to evaluate the association of various ecological microbial communities, such as soil, water and rhizosphere. Presented here is a protocol on how to use the WGCNA algorithm to analyze different co-occurrence networks that may occur in the microbial communities due to different ecological environments.

基于加权相关网络的不同栖息地根微生物群的差异

The root microbiome plays an important role in plant growth and environmental adaptation. Network analysis is an important tool for studying communities, ...