Name: plant growth and agrobacterium-mediated floral-dip transformation of the extremophyte schrenkiella parvula
Uploaded: 2019-01-07T14:00:00
Description: Watch this Scientific Journal Video about Plant Growth and Agrobacterium-mediated Floral-dip Transformation of the Extremophyte Schrenkiella parvula at JoVE.com

这项工作得到了国家科学基金会 mcb 1616827 奖的支持。

1. 植物生长
<ol><li>
种子灭菌 (可选)
	<ol>
<li>在双蒸馏水 (ddh2o)中制备50% 的漂白剂, 在50毫升管中制备1或2滴非离子洗涤剂 (见材料表)。将管反转多次, 混合溶液。 请注意:最好在具有紫外线灭菌表面的层流柜中进行种子灭菌15分钟。</li>
		<li>在 1.5 ml 管中加入漂白剂溶液, 加入 ~ 100–200 s. parvula 种子。充分混合, 让管坐5分钟。</li>
		<li>从试管中?...

<ol>
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植物的生理状态对转化效率有显著影响.利用健康而有活力的植物进行转化是成功转化的关键要求。与理想转化的健康植物相比, 水或光有压力的植物的花将更少 (图 1, 中心面板)。在低于 130μmol m-2 s-1 的光强下生长, 但植物往往更脆弱;这样的植物会导致更多的流产花后, 花浸水。帕鲁拉倾向于以高于塔利亚纳的速度...

在这个协议中, 我们描述了生长和建立稳定的转基因线的极端植物模型雪伦基耶拉帕维拉。有效的转化系统的可用性是任何多功能遗传模型的标志。在极端环境中茁壮成长的植物, 被称为极端植物, 为了解植物对环境压力的适应提供了重要资源。施伦基耶拉·帕弗拉(原thellungiella parvula和eutrema parvula) 就是这样一种极端植物模型, 具有不断扩展的基因组资源1,2, 3,4, 5.然而, 在已发表的研究中, 还没有报道 s . parvula 的转化协议。
帕拉维的基因组是布拉西卡科 (芥末科) 首次公布的极生植物基因组, 并与非极生植物模型拟南芥1进行了广泛的整体基因组联合。因此,对塔利亚纳和阴道的比较研究可以受益于在塔利亚纳进行的大量基因研究, 以便对阴道基因组是如何进化和调控的做出信息性假设以应对极端环境压力5,6,7。在已知的野生亲缘植物中,黄花是最耐盐的物种之一 (基于土壤 ncl ld50)。除了氯化钠耐受性外, 在高浓度对大多数植物有毒的情况下, s. parvula还存活并完成其生命周期.为了应对其自然栖息地普遍存在的非生物胁迫, 它进化了各种性状, 其中一些已在生化或生理水平8,9,10, 11个。
自2010年以来, 有400多种同行更新的出版物将s. parvula作为目标物种, 或将其用于与其他植物基因组的比较。然而, 通过更仔细地研究已经进行了哪些类型的研究, 可以确定一个明显的瓶颈。这些报告大多讨论了在未来研究中的潜在用途, 或将其用于比较基因组或系统学研究。由于没有为s. parvula制定概念验证转换协议, 尽管迄今为止已将最高质量的植物基因组之一组装和组装, 但该协议尚未用于功能基因组研究。注释成染色体级的假分子1。
农杆菌介导的花浸转化方法已成为在塔利亚纳中最广泛使用的产生植物基因的方法, 开发可复制的转化系统是其成功的关键因素。遗传模型12,13。然而, 并非所有的布拉西卡科物种都已被证明是成功地转化使用了为a.thaliana 开发的花浸法。特别是包括白斑的 Brassicaceae 系 ii 种一直对以植物浸渍为基础的转化方法14,15。
由于罗非鱼开花习性不确定, 加上叶片形态狭窄, 采用标准农杆菌介导的花浸转化方法具有挑战性。在这项研究中, 我们报告了我们为s. parvula的可重复转化而开发的修改后的协议。

<table border="0" cellpadding="0" cellspacing="0" style="width:1303px;" width="1302">
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	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:341px;">Agar</td>
			<td style="width:293px;">VWR International, Radnor, PA</td>
			<td style="width:127px;">90000-762</td>
			<td style="width:541px;">Bacto Agar Soldifying Agent, BD Diagnostics</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">B5 vitamins</td>
			<td>Sigma-Aldrich, St. Louis, MO</td>
			<td>G1019</td>
			<td>Gamborg&#8217;s Vitamin Solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:341px;">Desiccant</td>
			<td>W A Hammond Drierite, Xenia, OH</td>
			<td style="width:127px;">22005</td>
			<td>Indicating DRIERITE 6 mesh</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:341px;">Destination vector for plant transformation</td>
			<td>TAIR</td>
			<td style="width:127px;">Vector:6531113857</td>
			<td>pKGWFS7</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Electroporation cuvette</td>
			<td>USA Scientific</td>
			<td>9104-5050</td>
			<td>Electroporation cuvette, round cap, 0.2 cm gap</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Electroporator</td>
			<td>BIO-RAD Laboratories, Hercules, CA</td>
			<td>1652100</td>
			<td>MicroPulser Electroporator</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fertilizer beads</td>
			<td>Osmocote Garden, Marysville, OH</td>
			<td>N/A</td>
			<td>Osmocote Smart-Release Plant Food&#160;Flower &#38; Vegetable</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Gel extraction kit</td>
			<td>iNtRON Biotechnology, Boston, MA</td>
			<td>17289</td>
			<td>MEGAquick-spin Total fragment DNA purification kit</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Gentamicin</td>
			<td>Sigma-Aldrich, St. Louis, MO</td>
			<td>G1914-5G</td>
			<td>Gentamicin sulfate</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:341px;">Glufosinate-ammonium (11.3%) herbicide (BASTA)</td>
			<td>Bayer environmental science, Montvale, NJ</td>
			<td>N/A</td>
			<td>FINALE herbicide</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Kanamycin</td>
			<td>VWR International, Radnor, PA</td>
			<td>200004-444</td>
			<td>Kanamycin monosulfate</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MES</td>
			<td>Bioworld, Dublin, OH</td>
			<td>41320024-2</td>
			<td>MES, Free Acid</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MS salt</td>
			<td>MP Biomedicals, Santa Anna, CA</td>
			<td>092621822</td>
			<td>Hoagland&#39;s modified basal salt mixture</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">N6-benzylaminopurine (BA)&#160;</td>
			<td>Sigma-Aldrich, St. Louis, MO</td>
			<td>B3274</td>
			<td>6-Benzylaminopurine solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NaCl</td>
			<td>Sigma-Alrich</td>
			<td>S7653</td>
			<td>Sodium chloride</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Non-ionic detergent</td>
			<td>Sigma-Aldrich, St. Louis, MO</td>
			<td>9005-64-5</td>
			<td>TWEEN 20&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:341px;">Plasmid isolation kit</td>
			<td>Zymo Research, Irvine, CA</td>
			<td>D4036</td>
			<td>Zyppy Plasmid Kits</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Recombinase enzyme mix kit</td>
			<td>Life Technology</td>
			<td>11791-020</td>
			<td>Gateway LR Clonase II Enzyme mix</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Rifampicin</td>
			<td>Sigma-Aldrich, St. Louis, MO</td>
			<td>R3501-1G</td>
			<td>Rifampicin, powder, &#62;= 97% (HPLC)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Shaking incubator</td>
			<td>ThermoFisher Scientific, Waltham, MA</td>
			<td>SHKE4450</td>
			<td>MaxQ 4450 Benchtop Orbital Shakers</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Soil mix</td>
			<td>Sun Gro</td>
			<td>SUN239223328CFLP</td>
			<td>Sun Gro Metro-Mix 360 Grower Mix</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Spectinomycin</td>
			<td>VWR International, Radnor, PA</td>
			<td>IC15206705</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sterile 50ml conical tubes</td>
			<td>USA Scientific, Ocala, FL</td>
			<td>1500-1811</td>
			<td>50 ml conical screw cap tubes, copolymer, racks, sterile</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sucrose</td>
			<td>VWR International, Radnor, PA</td>
			<td>57-50-1</td>
			<td>Sucrose, ACS</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Surfactant solution</td>
			<td>Lehle seeds, Round Rock, TX</td>
			<td>VIS-02</td>
			<td>Silwet L-77</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Topoisomerase-based cloning kit</td>
			<td>Life Technologies, Carlsbad, CA</td>
			<td>K240020</td>
			<td>pENTR/D-TOPO Cloning Kit, with One Shot TOP10 Chemically Competent E. coli</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tryptone</td>
			<td>VWR International, Radnor, PA</td>
			<td>90000-282</td>
			<td>BD Bacto Tryptone, BD Biosciences</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Yeast Extract</td>
			<td>VWR International, Radnor, PA</td>
			<td>90000-722&#160;</td>
			<td>BD Bacto Yeast Extract, BD Biosciences</td>
		</tr>
	</tbody>
</table>

plant growth and agrobacterium-mediated floral-dip transformation of the extremophyte schrenkiella parvula

夏氏叶炎是一种适应各种非生物胁迫的极端植物, 包括多重离子毒性胁迫。尽管有高质量的基因组资源可用于研究植物如何适应环境压力, 但由于缺乏可行的转化系统, 其作为功能基因组学模型和工具的价值受到限制。在这个协议中, 我们报告如何产生稳定的转基因s.类似线使用农杆菌介导的花浸法。我们修改了用于罗非鱼的转化协议, 以考虑到罗非鱼的独特特征, 如不确定的开花习惯和高表观蜡含量的叶子。简单地说, 在种植前,将其在4°c 下分层5天。植物生长在14小时的光和10小时黑暗的光周期和 130μmol m-2-1 的光强, 在 22°c 至24°c。选择了8至9个具有多个花序的周龄植物进行改造。这些花序被浸入携带 mp90rk质粒的农杆菌 gv3101 的浸润溶液中。我们在三到四周的时间间隔内进行了两轮插花, 以提高转化效率。t1 种子在萌发前在容器中收集并干燥四周, 以筛选出候选转化线。对 basta 的抗性被用来筛选 t1 植物。我们在两个为期一周的工厂喷洒了三次基本服务溶液, 间隔为三天, 以减少假阳性。对存活的单个植物进行了基本程度的空投试验, 以确定真正的阳性转化物。转化效率为 0.033, 每繁殖 10, 000 种 t1 种子产生3-4 个转基因植株。

我们开发了一个转化协议, 可以在150天内收获 t 0 种子, 使用的是从 a. thaliana的花浸法修改的方法.图 1显示了时间线和s. parvula 植物的摘要, 它们代表了通过花浸执行转换的最佳阶段。我们选择了在萌发后60-80天内具有70-80 朵花的植物作为转化的目标阶段。在这一阶段, 少量先前存在的开放或受精卵和花在植物浸润之前被花<em...

Watch this Scientific Journal Video about Plant Growth and Agrobacterium-mediated Floral-dip Transformation of the Extremophyte Schrenkiella parvula at JoVE.com

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

植物生长与农杆菌介导的植物浸水转化

利用花浸法进行农杆菌介导的转化, 可以成功地利用这种方法, 创造出稳定的极端植物模型schrenkiella parvula的转基因线。考虑到不同的生长习性和四肢的生理特性, 我们提出了一种从这种方法中修改的方案。

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

这种方法是Schrenkiella parvula的可重复转化方案，可以帮助回答植物比较和功能基因领域的问题，特别是研究植物适应恶劣环境的研究。该技术能够研究施伦基拉帕武拉的常规功能，对适应多离子盐应力的极生植物进行检测。它还支持比较研究，也看其近亲，母亲父母阿里比多普西斯·塔利亚娜。将施伦基拉·帕尔武拉与阿里比多普西斯·塔利亚纳进行比较，可以深入了解基因调控和功能的差异，使施伦基埃拉帕武拉具有独特的适应能力。阿拉伯塔利亚纳的转型议定书已经确立。这种方法现在能够使用转基因施伦基拉帕武拉植物进行比较研究。一般来说，由于施伦基拉帕尔武拉植物的开花习惯和叶形态，利用植物浸渍进行有效转化以及选择真正的转化剂困难，因此对这种方法的新研究人员将难以进行学习。首先，使用湿牙签将大约 20 到 25 S.Parvula 种子转移到湿土上。然后在四摄氏度下分层，为期五至七天。分层后，将种子返回生长室7至10天。一旦所有的幼苗都发芽了，在四个角落和盆的中心只留下一个幼苗。在生长室中种植植物8至10周，直到它们开花。在转换当天，根据文本协议选择 S.Parvula 植物。使用钳子和小剪刀去除已经发展成硅的授粉花序。植物准备好后，准备一个农业细菌渗透溶液。在此之后，在农业细菌渗透溶液中浸入S.Parvula花样20秒。这里展示的是可用于转化的植物的最早阶段，只要在转化前去除已经发育的硅酸盐，可以使用花朵更多的较老植物。改造后，将花浸植物水平放在干净的托盘与圆顶覆盖植物。然后将植物放在黑暗的生长室一到两天。将植物返回直立位置，并转移到生长室。在下面的一周，监测浸花序。第一次花浸后十天，S.Parvula植物将继续生产新的花序和花香。第二轮花卉浸渍可以进行，以进一步改变新兴的花序。种植植物，直到种子成熟，收获种子约21周。在室温下，在装满干燥剂的密封容器中干燥种子两到三周。根据文本协议种植 T 一种子。种植植物，直到前两到三个真正的叶子发展。要进行抗除草剂的第一选择，稀释葡萄糖酸铵除草剂并喷洒在幼苗上。用圆顶盖住幼苗过夜。将托盘放回生长室，然后取下圆顶。等到除草剂选出大部分幼苗。要开始第二个选择，请确定使用 BASTA 溶液喷洒三到四次后存活下来的植物。在种植植物两到三周后，选择每个植物上最大的成熟叶。接下来用手指轻轻擦擦叶子的表面，去除蜡层。然后将稀释的BASA溶液滴在叶子上。使用 BASTA 溶液监测处理的叶子是否有枯萎迹象长达一周。最后选择叶子不受BASA滴影响的植物作进一步分析。在该协议中，S.Parvula使用花浸法对农业细菌进行转化。农业细菌的感染导致一些花流产。然而，S.Parvula植物在第一次花浸后继续生产新花，并可以再次转化。使用 BASTA 喷雾和跌落测试，可以准确识别真正的正转化剂。可减少使用 PCR 确认测试的样品数量。虽然这种方法类似于阿拉伯塔利亚纳的转化，但重要的是要记住施伦基拉·帕尔武拉的生长和形态差异。修改协议以反映这些差异对于成功转换至关重要。这项技术可以帮助研究人员探索基因组变异的进化，导致多种盐应力适应在极性植物模型，施伦基拉帕尔武拉。将这项技术与阿拉多皮斯·塔利亚纳（Aradopsis Thaliana）的功能信息相结合，该植物与施伦基埃拉·帕尔武拉密切相关。研究人员可以研究这两个物种之间的两种不同结构，在调控和功能上的变化。在转化过程中，在可 <3>性容器中包含所有农业细菌渗透溶液非常重要。消毒并安全地丢弃与农业细菌接触的所有材料，包括手套、宏移液器提示和纸巾。

Research

JoVE Journal

Genetics

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

A Robotic Platform for High-throughput Protoplast Isolation and Transformation

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

Genetic Manipulation of the Plant Pathogen Ustilago maydis to Study Fungal Biology and Plant Microbe Interactions

Genetic Manipulation of the Plant Pathogen Ustilago maydis to Study Fungal Biology and Plant Microbe Interactions

植物病原体的遗传操作<em&gt;玉米黑粉菌</em&gt;研究真菌生物学和植物微生物相互作用

Gene deletion plays an important role in the analysis of gene function. One of the most efficient methods to disrupt genes in a targeted manner is the ...

The Use of Induced Somatic Sector Analysis (ISSA) for Studying Genes and Promoters Involved in Wood Formation and Secondary Stem Development

The Use of Induced Somatic Sector Analysis ISSA for Studying Genes and Promoters Involved in Wood Formation and Secondary Stem Development

诱导体细胞行业分析（ISSA）的为研究木材形成和二次开发茎相关基因和推动者使用

Secondary stem growth in trees and associated wood formation are significant both from biological and commercial perspectives. However, relatively little ...

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

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

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 ...

Assessment of DNA Contamination in RNA Samples Based on Ribosomal DNA

基于核糖体 dna 的 RNA 样本 dna 污染评价

One method extensively used for the quantification of gene expression changes and transcript abundances is reverse-transcription quantitative real-time ...

Purification of High Molecular Weight Genomic DNA from Powdery Mildew for Long-Read Sequencing

超高分子量基因组DNA的分离纯化白粉病对长读测序

The powdery mildew fungi are a group of economically important fungal plant pathogens. Relatively little is known about the molecular biology and genetics ...

Optimization and Comparative Analysis of Plant Organellar DNA Enrichment Methods Suitable for Next-generation Sequencing

适用于下一代测序的植物细菌DNA富集方法的优化与比较分析

Plant organellar genomes contain large, repetitive elements that may undergo pairing or recombination to form complex structures and/or sub-genomic ...

Robust DNA Isolation and High-throughput Sequencing Library Construction for Herbarium Specimens

植物标本标本的鲁棒 DNA 分离与高通量测序库建设

Herbaria are an invaluable source of plant material that can be used in a variety of biological studies. The use of herbarium specimens is associated with ...

Exploring the Root Microbiome: Extracting Bacterial Community Data from the Soil, Rhizosphere, and Root Endosphere

从土壤、根际和根 Endosphere 提取细菌群落数据的根菌群探析

The intimate interaction between plant host and associated microorganisms is crucial in determining plant fitness, and can foster improved tolerance to ...

Discrimintion and Mapping of the Primary and Processed Transcripts in Maize Mitochondrion Using a Circular RT-PCR-based Strategy

使用基于循环 RT-PCR 的策略对玉米 Mitochondrion 中初级和已处理转录的区分和映射

In plant mitochondria, some steady-state transcripts have 5&#39; triphosphate derived from transcription initiation (primary transcripts), while the ...