Name: plant promoter analysis: identification and characterization of root nodule specific promoter in the common bean
Uploaded: 2017-12-23T12:30:00
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...

<ol>
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K., S&#225;nchez-L&#243;pez, R., Nava, N., Santana, O., C&#225;rdenas, L., Quinto, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RbohB+a+Phaseolus+vulgaris+NADPH+oxidase+gene,+enhances+symbiosome+number,+bacteroid+size,+and+nitrogen+fixation+in+nodules+and+impairs+mycorrhizal+colonization.">RbohB a Phaseolus vulgaris NADPH oxidase gene, enhances symbiosome number, bacteroid size, and nitrogen fixation in nodules and impairs mycorrhizal colonization.</a> New Phytol. 202 (3), 886-900 (2014).</li><li>Barraza, A., 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=Down-regulation+of+PvTRE1+enhances+nodule+biomass+and+bacteroid+number+in+the+common+bean.">Down-regulation of PvTRE1 enhances nodule biomass and bacteroid number in the common bean.</a> New Phytol. 197 (1), 194-206 (2013).</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=An+autophagy-related+kinase+is+essential+for+the+symbiotic+relationship+between+Phaseolus+vulgaris+and+both+rhizobia+and+arbuscular+mycorrhizal+fungi.">An autophagy-related kinase is essential for the symbiotic relationship between Phaseolus vulgaris and both rhizobia and arbuscular mycorrhizal fungi.</a> Plant Cell. 28 (9), 2326-2341 (2016).</li><li>Nanjareddy, K., 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=A+Legume+TOR+Protein+Kinase+Regulates+Rhizobium+Symbiosis+and+Is+Essential+for+Infection+and+Nodule+Development.">A Legume TOR Protein Kinase Regulates Rhizobium Symbiosis and Is Essential for Infection and Nodule Development.</a> Plant Physiol. 172 (3), 2002-2020 (2016).</li><li>Fr&#252;hling, M., Schr&#246;der, G., Hohnjec, N., P&#252;hler, A., Perlick, A. M., K&#252;ster, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+promoter+of+the+Vicia+faba+L.+gene+VfEnod12+encoding+an+early+nodulin+is+active+in+cortical+cells+and+nodule+primordia+of+transgenic+hairy+roots+of+Vicia+hirsuta+as+well+as+in+the+prefixing+zone+II+of+mature+transgenic+V.+hirsuta+root+nodules.">The promoter of the Vicia faba L. gene VfEnod12 encoding an early nodulin is active in cortical cells and nodule primordia of transgenic hairy roots of Vicia hirsuta as well as in the prefixing zone II of mature transgenic V. hirsuta root nodules.</a> Plant Science. 160 (1), 67-75 (2000).</li><li>Szabados, L., Ratet, P., Grunenberg, B., de Bruijn, F. 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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...

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>
			<td style="width:460px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">pNIN Reverse</td>
			<td style="width:253px;">CAT CTT CCT TCC ACT AAC TAA C</td>
			<td style="width:460px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">M13 Forward</td>
			<td>GTA AAA CGA CGG CCA G</td>
			<td style="width:460px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">M13 Reverse</td>
			<td>CAG GAA ACA GCT ATG AC</td>
			<td style="width:460px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">Name</td>
			<td style="width:253px;">Company</td>
			<td style="width:460px;">Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">REAGENTS</td>
			<td style="width:253px;"></td>
			<td style="width:460px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">pENTR/D-TOPO Cloning Kit</td>
			<td style="width:253px;">Invitrogen</td>
			<td style="width:460px;">K243520</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Gateway LR Clonase II Enzyme Mix&#160;</td>
			<td style="width:253px;">Invitrogen</td>
			<td style="width:460px;">11791100</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">pBGWFS7.0&#160;</td>
			<td style="width:253px;">Plant systems biology</td>
			<td style="width:460px;">https://gateway.psb.ugent.be/vector/show/pBGWFS7/search/index/</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Platinum Taq DNA Polymerase</td>
			<td style="width:253px;">ThermoFisher Scientific</td>
			<td style="width:460px;">10966018</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">DNeasy Plant Mini Kit</td>
			<td style="width:253px;">Qiagen</td>
			<td>69104</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">PureLink Quick Gel Extraction Kit</td>
			<td style="width:253px;">ThermoFisher Scientific</td>
			<td style="width:460px;">K210012</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">Platinum Pfx DNA Polymerase</td>
			<td style="width:253px;">Invitrogen</td>
			<td style="width:460px;">11708013</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:245px;">Certified Molecular Biology Agarose</td>
			<td style="width:253px;">Bio-Rad</td>
			<td>1613102</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:245px;">One Shot TOP10 Chemically Competent E. coli</td>
			<td style="width:253px;">Invitrogen</td>
			<td style="width:460px;">C404006</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">Nacl</td>
			<td style="width:253px;">Sigma-Aldrich</td>
			<td style="width:460px;">S7653</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">Tryptone</td>
			<td style="width:253px;">Sigma-Aldrich</td>
			<td style="width:460px;">T7293-250G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">Yeast extract</td>
			<td style="width:253px;">Sigma-Aldrich</td>
			<td style="width:460px;">Y1625-250G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">Bacteriological agar</td>
			<td style="width:253px;">Sigma-Aldrich</td>
			<td style="width:460px;">A5306-1KG</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">Kanamycin sulfate</td>
			<td style="width:253px;">Sigma-Aldrich</td>
			<td style="width:460px;">60615-25G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">Spectinomycin sulfate</td>
			<td style="width:253px;">Sigma-Aldrich</td>
			<td>PHR1441</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">Ethyl alcohol</td>
			<td style="width:253px;">Sigma-Aldrich</td>
			<td>E7023</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">Bacteriological peptone</td>
			<td style="width:253px;">Sigma-Aldrich</td>
			<td style="width:460px;">P0556</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">Calcium chloride</td>
			<td style="width:253px;">Sigma-Aldrich</td>
			<td style="width:460px;">C1016</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">Nalidixic acid</td>
			<td style="width:253px;">Sigma-Aldrich</td>
			<td style="width:460px;">N8878</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">Magnesium sulfate</td>
			<td style="width:253px;">Sigma-Aldrich</td>
			<td style="width:460px;">M7506</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">Gel loading solution</td>
			<td style="width:253px;">Sigma-Aldrich</td>
			<td style="width:460px;">G7654</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">Name</td>
			<td style="width:253px;">Company</td>
			<td style="width:460px;">Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">EQUIPMENT</td>
			<td style="width:253px;"></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">Thermocycler</td>
			<td style="width:253px;">Veriti Thermal Cycler</td>
			<td style="width:460px;">4375786</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">Centrifuge</td>
			<td style="width:253px;">Sigma</td>
			<td style="width:460px;">Sigma 1-14K</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">Gel documentation unit</td>
			<td style="width:253px;">Carestream&#160;</td>
			<td style="width:460px;">Gel Logic 212 PRO</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:245px;">MaxQ SHKE6000 6000 Shaking Incubator - 115VAC</td>
			<td style="width:253px;">Thermo scientific</td>
			<td style="width:460px;">EW-51708-70</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">Plant growth chamber</td>
			<td style="width:253px;">MRC</td>
			<td>PGI-550RH&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">Horizantal laminarair flow cabinate</td>
			<td style="width:253px;">Lumistell</td>
			<td style="width:460px;">LH-120</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">Fluorescent microscope</td>
			<td style="width:253px;">Leica&#160;</td>
			<td style="width:460px;">DM4500 B</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">Petridish</td>
			<td style="width:253px;">sym laboratorios</td>
			<td style="width:460px;">90X15</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">Scalpel Blade&#160;</td>
			<td style="width:253px;">Fisher scientific</td>
			<td style="width:460px;">53223</td>
			<td></td>
		</tr>
		<tr height="42">
			<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 g...

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 ...

Research

JoVE Journal

Biology

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

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

Identification of Post-translational Modifications of Plant Protein Complexes

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

Generation of Plasmid Vectors Expressing FLAG-tagged Proteins Under the Regulation of Human Elongation Factor-1ÃƒÅ½Ã‚Â± Promoter Using Gibson Assembly

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 Inducible System in Arabidopsis 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

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

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

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

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

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, ...

An Improved Chemotaxis Assay for the Rapid Identification of Rhizobacterial Chemoattractants in Root Exudates

一种用于快速鉴定根系分泌物中根细菌化学引诱剂的改进趋化性测定

Chemotaxis identification is very important for the research and application of rhizosphere growth-promoting bacteria. We established a straightforward ...

Isolation and Characterization of Intact Phycobilisome in Cyanobacteria

蓝藻中完整藻胆体的分离与表征

In cyanobacteria, phycobilisome is a vital antenna protein complex that harvests light and transfers energy to photosystem I and II for photochemistry. ...