Name: generating homo- and heterografts between watermelon and bottle gourd for the study of cold-responsive micrornas
Uploaded: 2018-11-20T12:30:00
Description: Watch this Scientific Journal Video about Generating Homo- and Heterografts Between Watermelon and Bottle Gourd for the Study of Cold-responsive MicroRNAs at JoVE.com

这项工作得到了国家自然科学基金 (31772191)、浙江省公益研究项目 (2017c32027)、浙江省植物育种重点科学项目 (2017C32027) 和国家项目计划的支持。支持一流的青年专业人员 (到 p. x.)。

1. 种子灭菌和萌发
<ol><li>对于表面灭菌, 将瓶装葫芦种子浸泡在500毫升的烧杯中, 在58°c 下充满水, 偶尔搅拌, 直到水温下降到40°c。</li>
	<li>同时, 将3公斤泥炭土放入尼龙袋中, 对其进行灭菌, 以 120°cse0.5 mpa 高压灭菌20分钟。</li>
	<li>在不搅拌的情况下, 将葫芦种子再浸泡4-5小时。<ol>
<li>一旦水达到室温, 用蒸馏水冲洗种子 2x-3倍。</li>
		<li>将多余的水排干, 让种子在 2 8°c 的纱布袋中发芽, 在黑暗中?...

<ol>
	<li>Schwarz, D., Rouphael, Y., Colla, G., Venema, J. 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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=miR395+is+a+general+component+of+the+sulfate+assimilation+regulatory+network+in+Arabidopsis.">miR395 is a general component of the sulfate assimilation regulatory network in Arabidopsis.</a> FEBS Letters. 586 (19), 3242-3248 (2012).</li><li>Ali, E. M., 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=Transmission+of+RNA+silencing+signal+through+grafting+confers+virus+resistance+from+transgenically+silenced+tobacco+rootstocks+to+non-transgenic+tomato+and+tobacco+scions.">Transmission of RNA silencing signal through grafting confers virus resistance from transgenically silenced tobacco rootstocks to non-transgenic tomato and tobacco scions.</a> Journal of Plant Biochemistry and Biotechnology. 25 (3), 245-252 (2016).</li><li>Li, Y. S., Li, C. 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在该方案中, 我们详细介绍了一种高效、可重复的方法, 使西瓜与葫芦之间的同质和异种移植物。这种方法不需要特定的设备, 操作起来非常容易, 通常嫁接的存活率非常高。该方法还可用于制作其他黄瓜的嫁接物, 如西瓜、黄瓜和南瓜之间的嫁接物。
值得注意的是, 根茎和接穗的相对大小 (年龄) 对于成功嫁接至关重要 ( 《议定书》第2.2 步)。我们观察到, 如果所使?...

长期以来, 嫁接一直被用作提高作物产量和耐受生物和非生物胁迫 1,2,3的农业技术。在异质嫁接系统中, 精英根系可以提高植物的水分和养分吸收, 增强对土壤病原体的抵抗力, 并限制金属毒性 4,5的负面影响, 这可能会增加嫁接增长活力和提高对环境压力的耐受性。在许多情况下, 异种嫁接也会影响园艺植物中的果实品质, 从而改善水果风味, 增加与健康相关的化合物6,7的含量。研究发现, 植物激素、rna、肽和蛋白质在根茎和接穗之间的远距离转移是调节接穗植物生长发育的一个基本机制 8,9 ,10。接枝在与环境适应有关的远距离信号和运输研究中得到了广泛的应用。接枝实验对于在接收组织或血管液中明确检测传输分子以及由于信号传输而激活或抑制分子目标的作用特别大。
据报道, 非编码 rna 是一种在细胞中发挥重要调控功能的大一类 rna, 它在促进植物适应非生物胁迫方面发挥了作用.mirna 是大约 20-24 nt 的内生小非编码 rna. 研究揭示了 mirna 在植物活动各个方面的调控作用, 如幼苗生长、侧根形成14、15、16、营养吸收, 硫酸盐代谢和稳态 17,以及对生物和非生物胁迫的反应18。近年来, mirna 及其靶向基因的表达与异种嫁接黄瓜幼苗的耐盐胁迫性有关.在葡萄的种间移植中, mirna 表达对干旱胁迫的反应被发现是遗传依赖性的 20。
高通量测序技术的快速发展和成本的降低, 为研究农艺植物中的 mirna 规律提供了良好的契机。西瓜 (瓜兰特 [thunb.]曼斯夫) 是全世界种植的一种重要的葫芦作物, 容易受到低温的影响。瓶装葫芦是一种更具气候弹性的黄瓜, 农民通常用西瓜移植。本研究的主要目的是建立一种标准、高效、简便的西瓜之间异种移植方法.曼斯夫)和瓶装葫芦 (紫菜 [molina] standl).该协议还提供了一个详细的实验方案和分析程序, 用于研究接枝后 mirna 表达的调节, 这对于揭示异质接枝优势背后的机制是有用的。
本研究使用的植物材料包括西瓜品种和葫芦林。西瓜品种是一种产量高、但容易受到低温影响的商业品种。瓶装葫芦是一种很受欢迎的西瓜、黄瓜和葫芦嫁接的根茎, 因为它具有良好的耐低温能力.

<table>
	<tbody>
		<tr>
			<td>TRIzol Reagent</td>
			<td>Invitrogen</td>
			<td>15596026</td>
			<td></td>
		</tr>
		<tr>
			<td>RNA-free DNase I</td>
			<td>Takara</td>
			<td>D2270A</td>
			<td></td>
		</tr>
		<tr>
			<td>Truseq Small RNA sample prep Kit</td>
			<td>Illumina</td>
			<td>RS-200-0012</td>
			<td></td>
		</tr>
		<tr>
			<td>2100 Bionalyser</td>
			<td>Agilent</td>
			<td>5067</td>
			<td></td>
		</tr>
		<tr>
			<td>DNA Polymerase</td>
			<td>Thermo Fisher Scientific</td>
			<td>F530S</td>
			<td></td>
		</tr>
		<tr>
			<td>UEA sRNA workbench 2.4-plant version&#160;(software)</td>
			<td>NA</td>
			<td>NA</td>
			<td>http://srna-workbench.cmp.uea.ac.uk/</td>
		</tr>
		<tr>
			<td>Rfam 11.0 database&#160;(website)</td>
			<td>NA</td>
			<td>NA</td>
			<td>http://rfam.janelia.org</td>
		</tr>
		<tr>
			<td>miRBase 22.0&#160;(website)</td>
			<td>NA</td>
			<td>NA</td>
			<td>http://www.mirbase.org/</td>
		</tr>
		<tr>
			<td>MIREAP(software)</td>
			<td>NA</td>
			<td>NA</td>
			<td>https://sourceforge.net/projects/mireap/</td>
		</tr>
		<tr>
			<td>TargetFinder&#160;(software)</td>
			<td>NA</td>
			<td>NA</td>
			<td>http://targetfinder.org/</td>
		</tr>
	</tbody>
</table>

generating homo- and heterografts between watermelon and bottle gourd for the study of cold-responsive micrornas

微 rna (mirna) 是大约20-24 特的内生小非编码 rna, 已知在植物发育和适应中发挥着重要作用。有越来越多的证据表明, 某些 mirna 的表达在嫁接时发生了变化, 这是农民通常用来提高作物对生物和非生物胁迫能力的农业做法。与包括西瓜在内的许多其他主要黄瓜相比, 瓶葫芦是一种具有固有气候抵御能力的作物, 使其成为西瓜中使用最广泛的根茎之一。最近高通量测序技术的发展为研究冷反应 mirna 及其对异种移植优势的贡献提供了巨大的机会;然而, 适当的实验程序是实现这一目标的先决条件。在这里, 我们提出了一个详细的协议, 有效地生成同异种之间的冷易感西瓜和耐寒瓶葫芦, 除了组织取样, 数据生成和数据分析的方法。所提出的方法也适用于其他植物移植系统, 用于检测各种环境压力下的 mirna 法规, 如热、干旱和盐度。

<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/58242/58242fig2.jpg"> 图 2: 在室温和冷应力条件下各种移植物的表型.(a) 该面板显示在室温下的同型和异种嫁接幼苗作为对照。(b) 该面板显示经过48小时的冷处理后的同型和异种嫁接幼苗。<a href="https://www.jove.com/files/ftp_upload/58242/58242fig2large.jpg" target="_blank">请点击这里查看此图的较大版本.</a></p...

Watch this Scientific Journal Video about Generating Homo- and Heterografts Between Watermelon and Bottle Gourd for the Study of Cold-responsive MicroRNAs at JoVE.com

Generating Homo- and Heterografts Between Watermelon and Bottle Gourd for the Study of Cold-responsive MicroRNAs

西瓜与瓶装葫芦之间的同源和异种移植在冷反应微 rna 研究中的应用

本文提出了一个详细的方案, 有效地制作西瓜和葫芦之间的同质和异种移植, 除了组织取样、数据生成和数据分析的方法, 用于研究冷反应微 rna。

MicroRNAs (miRNAs) are endogenous small non-coding RNAs of about 20 - 24 nt, known to play important roles in plant development and adaptation. There is ...

这种方法可以帮助回答miRNA对植物嫁接中压力能力的反应中的关键问题，例如在西瓜瓶葫芦嫁接中，miRNA在冷应力下是如何调节的。该技术的主要优点是，它是高效和可重复的方法，使同质和异质。它不需要特定的设备，它很容易执行，通常导致非常高的嫁接存活率。通过这种方法，可以深入了解西瓜瓶葫芦嫁接系统中miRNA对冷应力的反应。它也可以应用于其他现代生物，以揭示本地和长距离miRNA运输的机制。这种方法的视觉演示至关重要，因为嫁接步骤很难学习。这一步需要高级技能，是嫁接植物生存的关键。首先，将瓶葫芦种子浸泡在500毫升的烧杯中，水为58摄氏度。偶尔搅拌种子，直到水温降至40摄氏度。当水冷却时，将三公斤泥炭土放入尼龙袋中，然后进行自动处理。一旦水达到室温，用蒸馏水冲洗种子两到三次，然后排干多余的水。让种子在28摄氏度的纱布袋中发芽，在黑暗的生长室。发芽后，将种子播种到装满灭菌泥炭土壤的塑料锅中。当瓶葫芦幼苗发展出两个扁平的瓜子，用西瓜种子重复这个过程。在生长室中种植瓶葫芦和西瓜苗。下午每天给幼苗加一次水。接下来，将西瓜幼苗的低小三厘米切成两到三厘米。切在一边的瓶子葫芦幼苗的顶部， 就在上面的鳕子。然后用牙签在修剪过的瓶子葫芦幼苗的顶部打个洞。将修剪过的西瓜苗插入孔中，使异质。在此之后，使用前面描述的方法准备同质。首先，将嫁接的幼苗包裹在透明聚乙烯袋中，以保持较高的湿度。然后在生长室中保持包裹的幼苗七天。第七天后，取出袋子，让植物在相同的条件下生长7至10天。将幼苗分成两组，一组用于冷处理，另一组用于控制。将控制幼苗返回到相同的环境条件。将经过冷处理的幼苗放入设定为摄氏六度的生长室中，其暗照条件与对照组相同。48小时后，从嫁接中留下丝骨和根茎的样品。立即将样品冷冻在液氮中，并将其储存在零下70摄氏度。将冷冻样品转移到液氮中的两毫升微离心管中。在每个样品管中加入一个不锈钢珠，将组织均匀化为细粉。对于每个嫁接组合，从 10 株幼苗中提取等量的地面样品，并混合在 10 毫升离心管中。加入适当量的盐酸钠试剂。接下来，在37摄氏度下每毫升加入无RNADNase1至150个单位，一小时。然后确定微胶囊电泳系统的总RNA数量。在此之后，解冻库规范化试剂和适配器。然后用五个质和三个质适配器将小 RNase 开，并进行和纯化。按照制造商的准则，反向转录五个原和三个原色连接小 RNase。接下来，按照制造商的协议执行 PCR 扩增。然后使用微血管电泳系统确保RNA完整性数大于7。在微胶囊电泳系统上加载RNA库的一微升，以确保RNA完整性数大于7。最后，对高通量测序仪器上的小型RNA库进行测序。利用本协议，获得98%的嫁接成活率和室温和冷应力条件的表型。24种核苷酸的sRNA在所有嫁接组合中是最大的sRNA类，无论温度处理如何。经过48小时的冷处理，30个和268个微RNA分别上下调节。相反，在异质的叶子中，在根茎的叶子中，分别有31个和12个微RNA被向上和向下调节。在西瓜-西瓜同种，64和83微RNA分别向上和向下调节。这表明异质引起微RNA表达的深刻重新编程。在尝试此过程时，重要的是要记住女儿茎的相对大小和年龄，而 sion 对于成功移植至关重要。按照这个程序，可以执行其他方法，如lncRNA测序，蛋白质组分析，以研究lncRNA和蛋白质的调控和编码系统在嫁接系统中。该技术开发后，为植物非生物耐受性领域的研究铺平了道路，探索了库库比赛及以后植物嫁接优势的机制。不要忘记，使用沙尼丁盐酸可能是极其危险的，在执行此程序时应始终采取适当的预防措施。

Research

JoVE Journal

Environment

Transcript and Metabolite Profiling for the Evaluation of Tobacco Tree and Poplar as Feedstock for the Bio-based Industry

The global demand for food, feed, energy, and water poses extraordinary challenges for future generations. It is evident that robust platforms for the ...

科研

环境科学

The Use of High-resolution Infrared Thermography (HRIT) for the Study of Ice Nucleation and Ice Propagation in Plants

The Use of High-resolution Infrared Thermography HRIT for the Study of Ice Nucleation and Ice Propagation in Plants

高分辨率红外热成像（HRIT）的冰核和冰传播的植物研究中的应用

Freezing events that occur when plants are actively growing can be a lethal event, particularly if the plant has no freezing tolerance. Such frost events ...

Empirical, Metagenomic, and Computational Techniques Illuminate the Mechanisms by which Fungicides Compromise Bee Health

经验、基因和计算技术阐明了杀菌剂危害蜜蜂健康的机制

Growers often use fungicide sprays during bloom to protect crops against disease, which exposes bees to fungicide residues. Although considered ...

Protocol for Microplastics Sampling on the Sea Surface and Sample Analysis

协议塑料微粒采样的海面和样品分析

Microplastic pollution in the marine environment is a scientific topic that has received increasing attention over the last decade. The majority of ...

Extraction of Organochlorine Pesticides from Plastic Pellets and Plastic Type Analysis

从塑料颗粒中提取有机氯农药和塑料类型分析

Plastic resin pellets, categorized as microplastics (&#8804;5 mm in diameter), are small granules that can be unintentionally released to the environment ...

Experimental Column Setup for Studying Anaerobic Biogeochemical Interactions Between Iron (Oxy)Hydroxides, Trace Elements, and Bacteria

Experimental Column Setup for Studying Anaerobic Biogeochemical Interactions Between Iron OxyHydroxides, Trace Elements, and Bacteria

用于研究铁 (氧) 氢氧化物、微量元素和细菌之间厌氧生物地球化学相互作用的实验柱装置

Fate and speciation of trace elements (TEs), such as arsenic (As) and mercury (Hg), in aquifers are closely related to physio-chemical conditions, such as ...

Ecosystem Fabrication (EcoFAB) Protocols for The Construction of Laboratory Ecosystems Designed to Study Plant-microbe Interactions

Ecosystem Fabrication EcoFAB Protocols for The Construction of Laboratory Ecosystems Designed to Study Plant-microbe Interactions

用于研究植物-微生物相互作用的实验室生态系统制造 (EcoFAB) 协议

Beneficial plant-microbe interactions offer a sustainable biological solution with the potential to boost low-input food and bioenergy production. A ...

Experimental Study of the Relationship Between Particle Size and Methane Sorption Capacity in Shale

页岩中粒径与甲烷吸附能力关系的实验研究

The amount of adsorbed shale gas is a key parameter used in shale gas resource evaluation and target area selection, and it is also an important standard ...

Harvesting Venom Toxins from Assassin Bugs and Other Heteropteran Insects

从刺客臭虫和其他 Heteropteran 昆虫身上采集毒液毒素

Heteropteran insects such as assassin bugs (Reduviidae) and giant water bugs (Belostomatidae) descended from a common predaceous and venomous ancestor, ...

Choice and No-Choice Bioassays to Study the Pupation Preference and Emergence Success of Ectropis grisescens

Choice and No-Choice Bioassays to Study the Pupation Preference and Emergence Success of Ectropis grisescens

选择与无选择生物鉴定研究蛾 grisescens的化蛹偏好及出现的成功

Many insects live above the ground as larvae and adults and as pupate below the ground. Compared to the above-ground stages of their life cycles, less ...