Name: development of targeting induced local lesions in genomes (tilling) populations in small grain crops by ethyl methanesulfonate mutagenesis
Uploaded: 2019-07-16T13:00:00
Description: Watch this Scientific Journal Video about Development of Targeting Induced Local Lesions IN Genomes TILLING Populations in Small Grain Crops by Ethyl Methanesulfonate Mutagenesis at JoVE.com

这项工作得到了美国农业部国家粮食和农业研究所、Hatch项目1016879和马里兰州农业实验站通过MAES授权号2956952的支持。

1. 有效诱变的剂量曲线的准备
<ol><li>将100粒具有基因型感兴趣的种子浸泡在6个250 mL玻璃瓶中(每个烧瓶100个),含有50 mL的蒸馏水。在室温 (RT) 下,在 100 rpm 下摇动 8 小时,以便种子浸渍。</li>
	<li>在烟气罩中,通过在蒸馏水中分别溶解 0.167、0.249、0.331、0.415 和 0.498 mMS 溶液,制备 50 mL 的 0.4%、0.6%、0.8%、1.0% 和 1.2%(w/v)乙酸甲酸酯 (EMS) 溶液。 注: EMS 在 RT 上是液体,密度为 1.206 g/mL。 注意:在处理 EMS 时,?...

<ol>
	<li>McCallum, C. M., Comai, L., Greene, E. A., Henikoff, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeted+screening+for+induced+mutations.">Targeted screening for induced mutations.</a> Nature Biotechnology. 18 (4), 455-457 (2000).</li><li>Bentley, A., MacLennan, B., Calvo, J., Dearolf, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeted+Recovery+of+Mutations+in+Drosophila.">Targeted Recovery of Mutations in Drosophila.</a> Genetics. 156 (3), 1169-1173 (2000).</li><li>Tsai, H., 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=Discovery+of+Rare+Mutations+in+Populations:+TILLING+by+Sequencing.">Discovery of Rare Mutations in Populations: TILLING by Sequencing.</a> Plant Physiology. 156 (3), 1257-1268 (2011).</li><li>Caldwell, D. 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=A+structured+mutant+population+for+forward+and+reverse+genetics+in+Barley+(Hordeum+vulgare+L.).">A structured mutant population for forward and reverse genetics in Barley (Hordeum vulgare L.).</a> The Plant Journal. 40 (1), 143-150 (2004).</li><li>Hazard, B., 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=Induced+Mutations+in+the+Starch+Branching+Enzyme+II+(+SBEII+)+Genes+Increase+Amylose+and+Resistant+Starch+Content+in+Durum+Wheat.">Induced Mutations in the Starch Branching Enzyme II ( SBEII ) Genes Increase Amylose and Resistant Starch Content in Durum Wheat.</a> Crop Science. 52 (4), 1754-1766 (2012).</li><li>Rawat, N., 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+diploid+wheat+TILLING+resource+for+wheat+functional+genomics.">A diploid wheat TILLING resource for wheat functional genomics.</a> BMC Plant Biology. 12, 205 (2012).</li><li>Rawat, N., 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=TILL-D:+An+Aegilops+tauschii+TILLING+Resource+for+Wheat+Improvement.">TILL-D: An Aegilops tauschii TILLING Resource for Wheat Improvement.</a> Frontiers in Plant Science. 9, (2018).</li><li>Rawat, N., 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=Wheat+Fhb1+encodes+a+chimeric+lectin+with+agglutinin+domains+and+a+pore-forming+toxin-like+domain+conferring+resistance+to+Fusarium+head+blight.">Wheat Fhb1 encodes a chimeric lectin with agglutinin domains and a pore-forming toxin-like domain conferring resistance to Fusarium head blight.</a> Nature Genetics. 48 (12), 1576-1580 (2016).</li><li>Kippes, N., Chen, A., Zhang, X., Lukaszewski, A. J., Dubcovsky, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+and+characterization+of+a+spring+hexaploid+wheat+line+with+no+functional+VRN2+genes.">Development and characterization of a spring hexaploid wheat line with no functional VRN2 genes.</a> Theoretical and Applied Genetics. 129 (7), 1417-1428 (2016).</li><li>Greene, E. 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=Spectrum+of+Chemically+Induced+Mutations+From+a+Large-Scale+Reverse-Genetic+Screen+in+Arabidopsis.">Spectrum of Chemically Induced Mutations From a Large-Scale Reverse-Genetic Screen in Arabidopsis.</a> Genetics. 164 (2), 731-740 (2003).</li><li>Harwood, W. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advances+and+remaining+challenges+in+the+transformation+of+barley+and+wheat.">Advances and remaining challenges in the transformation of barley and wheat.</a> Journal of Experimental Botany. 63 (5), 1791-1798 (2012).</li><li>Henikoff, S., Comai, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-Nucleotide+Mutations+for+Plant+Functional+Genomics.">Single-Nucleotide Mutations for Plant Functional Genomics.</a> Annual Review of Plant Biology. 54 (1), 375-401 (2003).</li><li>Uauy, C., 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+modified+TILLING+approach+to+detect+induced+mutations+in+tetraploid+and+hexaploid+wheat.">A modified TILLING approach to detect induced mutations in tetraploid and hexaploid wheat.</a> BMC Plant Biology. 9 (1), 115 (2009).</li><li>Uauy, C., Wulff, B. B. H., Dubcovsky, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Combining+Traditional+Mutagenesis+with+New+High-Throughput+Sequencing+and+Genome+Editing+to+Reveal+Hidden+Variation+in+Polyploid+Wheat.">Combining Traditional Mutagenesis with New High-Throughput Sequencing and Genome Editing to Reveal Hidden Variation in Polyploid Wheat.</a> Annual Review of Genetics. 51 (1), 435-454 (2017).</li><li>Li, 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=The+Sequences+of+1504+Mutants+in+the+Model+Rice+Variety+Kitaake+Facilitate+Rapid+Functional+Genomic+Studies.">The Sequences of 1504 Mutants in the Model Rice Variety Kitaake Facilitate Rapid Functional Genomic Studies.</a> The Plant Cell. 29 (6), 1218-1231 (2017).</li><li>Jiao, Y., 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+Sorghum+Mutant+Resource+as+an+Efficient+Platform+for+Gene+Discovery+in+Grasses.">A Sorghum Mutant Resource as an Efficient Platform for Gene Discovery in Grasses.</a> The Plant Cell. 28 (7), 1551-1562 (2016).</li><li>Krasileva, K. V., 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=Uncovering+hidden+variation+in+polyploid+wheat.">Uncovering hidden variation in polyploid wheat.</a> Proceedings of the National Academy of Sciences. , 201619268 (2017).</li><li>Dong, C., Dalton-Morgan, J., Vincent, K., Sharp, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Modified+TILLING+Method+for+Wheat+Breeding.">A Modified TILLING Method for Wheat Breeding.</a> The Plant Genome. 2 (1), 39-47 (2009).</li><li>Till, B. J., Zerr, T., Comai, L., Henikoff, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+protocol+for+TILLING+and+Ecotilling+in+plants+and+animals.">A protocol for TILLING and Ecotilling in plants and animals.</a> Nature Protocols. 1 (5), 2465-2477 (2006).</li><li>Wu, J. -. L., 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=Chemical-+and+Irradiation-induced+Mutants+of+Indica+Rice+IR64+for+Forward+and+Reverse+Genetics.">Chemical- and Irradiation-induced Mutants of Indica Rice IR64 for Forward and Reverse Genetics.</a> Plant Molecular Biology. 59 (1), 85-97 (2005).</li><li>Feldman, M., Levy, A. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome+Evolution+Due+to+Allopolyploidization+in+Wheat.">Genome Evolution Due to Allopolyploidization in Wheat.</a> Genetics. 192 (3), 763-774 (2012).</li><li>Comai, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+advantages+and+disadvantages+of+being+polyploid.">The advantages and disadvantages of being polyploid.</a> Nature Reviews Genetics. 6 (11), 836-846 (2005).</li><li>Guo, H., 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=Development+of+a+High-Efficient+Mutation+Resource+with+Phenotypic+Variation+in+Hexaploid+Winter+Wheat+and+Identification+of+Novel+Alleles+in+the+TaAGP.L-B1+Gene.">Development of a High-Efficient Mutation Resource with Phenotypic Variation in Hexaploid Winter Wheat and Identification of Novel Alleles in the TaAGP.L-B1 Gene.</a> Frontiers in Plant Science. 8, (2017).</li><li>Rakszegi, 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=Diversity+of+agronomic+and+morphological+traits+in+a+mutant+population+of+bread+wheat+studied+in+the+Healthgrain+program.">Diversity of agronomic and morphological traits in a mutant population of bread wheat studied in the Healthgrain program.</a> Euphytica. 174 (3), 409-421 (2010).</li><li>Tsai, H., Ngo, K., Lieberman, M., Missirian, V., Comai, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tilling+by+Sequencing.">Tilling by Sequencing.</a> Plant Functional Genomics: Methods and Protocols. , 359-380 (2015).</li></ol>

TILLING是一种非常有价值的反向遗传学工具,用于基因验证,特别是对于基于转化的方法存在严重瓶颈的小颗粒。培养高突变频率的诱变种群是进行功能基因组学研究的关键步骤之一。发展强健的TILLING种群的最重要步骤是确定EMS的最佳浓度。M1中40%-60%的存活率是小麦和大麦4、6、18EMS诱变效果的良好指标。</sup...

基因组中的点突变可以为研究人员提供许多有用的用途。根据它们的性质和位置,这些突变可用于将功能分配给基因,甚至不同感兴趣的蛋白质域。另一方面,作为新基因变异的来源,可以使用表型屏筛选来选择有用的突变,并进一步用于作物改良。TILLING是一个强大的反向遗传学工具,包括化学诱变和检测靶基因中的序列变异。最早在阿拉伯拟南芥1和果蝇黑色素2中开发,TILLING种群已被开发和用于许多小谷物作物,如六倍体面包小麦(三叶草)3,大麦 (Ordeum 庸)4, 四倍体杜鲁姆小麦 (T. 三氯球菌杜鲁姆)5,双发化小麦 (T.单球体)6和"D" 基因组后代小麦Aegilops tauschii7.这些资源已被用来验证基因在调节非生物和生物应激性8,调节开花时间9,并开发营养优越的作物品种5的作用。
除使用烷基化诱变诱变剂(如乙酸甲酸酯(EMS)、阿齐德钠、N-甲基-N-硝酸钠(MNU)和甲基甲酸甲酸酯(MMS)外,其优越性优于其他反向遗传学工具,原因有若干原因。首先,诱变可以在几乎任何物种或植物10品种上进行,并且独立于转化瓶颈,在小谷物11的情况下尤其具有挑战性。其次,除了产生其他基因验证方法可以获得的敲除突变外,还可以诱导一系列误感和拼接突变,从而可以分辨感兴趣的蛋白质的单个域的功能。此外,TILLING生成整个基因组中不朽的突变集合;因此,单个种群可用于多个基因的功能验证。相反,其他反向遗传学工具只生成研究13下的基因特有的资源。通过TILLING识别的有用突变可以用于育种目的,不受调控,不像基因编辑,其非转基因分类在许多国家仍然不确定。这变得特别相关的小谷物,是国际贸易14。
TILLING是一种简单而有效的基因验证策略,需要开发诱变种群以研究感兴趣的基因。发展有效的诱变种群是确定基于TILLING的基因验证研究效率的关键。总体突变频率较低的一个蒂林种群表明,必须对不切实际的大种群进行所需的突变筛查,而高诱变浓度会导致种群死亡率高,且数量不足。诱变个体。一旦形成良好的种群,有多种方法来检测感兴趣的基因突变,平台的选择取决于实验规模和资源的可用性。全基因组测序和外兆体测序已被用来描述具有小基因组15、16的植物中蒂林种群的所有突变。两个TILLING种群的外泄序列测序已在面包和杜鲁姆小麦中进行,供公众识别理想的突变和订购突变的感兴趣线17。就理想突变的可用性而言,它是一个伟大的公共资源;然而,在基因验证研究中,野生型线应具有感兴趣的候选基因。不幸的是,对整个TILLING种群的外在体进行测序,以便在另一个背景下对几个候选基因进行反向遗传学验证,仍然是成本高昂的。Amplicon测序和基于Cel-1的检测已用于检测小麦靶向种群的突变,Cel-1测定更简单,无需计算知识,特别适用于验证少数具有基本基因的基因实验室设备6,18 。
在本文中,介绍了发展良好蒂林种群的方法,包括制备剂量曲线、突变种群的诱变和维持,以及使用基于PCR的Cel-1测定对突变种群进行筛查.该协议已经成功地实施,开发和利用三聚体、三聚体6、大麦、Aegilops tauchii和几个别人。其中包括这些方法的明确细节,以及有用的提示,这将有助于研究人员发展TILLING种群,使用EMS作为诱变任何小谷物植物的选择。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>96 well 1.1 ml microtubes in microracks</td>
			<td>National Scientific</td>
			<td>TN0946-08R</td>
			<td>For collecting leaf tissues</td>
		</tr>
		<tr>
			<td>Agarose I biotechnology grade</td>
			<td>VWR</td>
			<td>0710-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>Biosprint 96 DNA Plant Kit</td>
			<td>Qiagen</td>
			<td>941558</td>
			<td>Kit for DNA extraction</td>
		</tr>
		<tr>
			<td>Cel-1 endonuclease</td>
			<td>Extracted as described by Till et al 2006</td>
			<td></td>
			<td>Single strand specific endonuclease</td>
		</tr>
		<tr>
			<td>Centrifuge 5430 R</td>
			<td>Eppendorf</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Ethyl methanesulfonate</td>
			<td>Sigma Aldrich</td>
			<td>M-0880-25G</td>
			<td>EMS, Chemical mutagen</td>
		</tr>
		<tr>
			<td>Freeze Dry/Shell freeze system</td>
			<td>Labconco</td>
			<td></td>
			<td>For lyophilization of leaf tissue</td>
		</tr>
		<tr>
			<td>Kingfisher Flex purification system</td>
			<td>Thermo fisher scientific</td>
			<td>5400610</td>
			<td>High throughput DNA extraction robot</td>
		</tr>
		<tr>
			<td>My Taq DNA Polymerase</td>
			<td>Bioline</td>
			<td>BIO-21107</td>
			<td></td>
		</tr>
		<tr>
			<td>Nuclease free water</td>
			<td>Sigma aldrich</td>
			<td>W4502-1L</td>
			<td></td>
		</tr>
		<tr>
			<td>NuGenius gel imaging system</td>
			<td>Syngene</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Orbit Environ-shaker</td>
			<td>Lab-line</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>SPECTROstar Nano</td>
			<td>BMG LABTECH</td>
			<td></td>
			<td>Nano drop for DNA quantification</td>
		</tr>
		<tr>
			<td>T100 Thermal cycler</td>
			<td>BIO-RAD</td>
			<td>1861096</td>
			<td></td>
		</tr>
	</tbody>
</table>

development of targeting induced local lesions in genomes (tilling) populations in small grain crops by ethyl methanesulfonate mutagenesis

靶向诱导局部病变在基因组(TILLING)是一个强大的反向遗传学工具,包括化学诱变和检测靶基因的序列变异。TILLING 是一种非常有价值的功能基因组学工具,用于基因验证,特别是在基于转化的方法存在严重局限性的小颗粒中。培养一个强大的诱变种群是确定基于TILLING的基因验证研究的效率的关键。总体突变频率较低的一个蒂林种群表明,必须筛选数量庞大的人群以找到所需的突变,而高诱变浓度会导致种群死亡率高,导致数量不足诱变个体的数量。一旦形成有效种群,有多种方法来检测感兴趣的基因突变,平台的选择取决于实验规模和资源的可用性。Cel-1测定法和基于甘蔗的凝胶基方法用于突变物鉴定,方便、可重复,且平台资源密集度较低。它具有优势,因为它简单,不需要计算知识,并且特别适用于使用基本实验室设备验证少量基因。本文介绍了良好的蒂林种群的发育方法,包括制备剂量曲线、突变种群的诱变和维持,以及使用基于PCR的Cel-1测定对突变种群进行筛查.

图2显示了六倍体面包小麦品种Jagger、二倍体小麦三聚体单球体6的剂量曲线,以及小麦Aegilops tauschii7的基因组供体。期望50%生存率的EMS剂量分别为T.单孔、Ae.tauschii和T.aestivum的0.25%、0.6%和0.7%。 六倍体小麦的EMS容差较高,是由于其基因组缓冲能力。然而,尽管两者都是二倍体,EMS对T.单...

Watch this Scientific Journal Video about Development of Targeting Induced Local Lesions IN Genomes TILLING Populations in Small Grain Crops by Ethyl Methanesulfonate Mutagenesis at JoVE.com

Development of Targeting Induced Local Lesions IN Genomes TILLING Populations in Small Grain Crops by Ethyl Methanesulfonate Mutagenesis

描述是使用乙基甲酸酯 (EMS) 作为诱变剂在小谷物作物中开发靶向诱导局部病变(TILLING)人群的协议。还提供了使用Cel-1测定突变检测的协议。

通过乙基甲烷硫素突变在小谷物作物中靶向诱导局部病变(TILLING)种群的发展

Targeting Induced Local Lesions IN Genomes (TILLING) is a powerful reverse genetics tool that includes chemical mutagenesis and detection of sequence ...

该协议详细介绍了在小谷物作物中通过EMS突变发生具有高突变频率的强健的耕种种群的发展。耕种种群可用于功能基因组学，以及小谷物作物的遗传空间基因发现。使用这种技术开发的直到丁体种群具有高突变频率，此协议可应用于任何基因型。基于 Cel-1 测定的突变检测可以使用基本的实验室设备进行。最重要的步骤是确定最佳 EMS 浓度。因此， EMS 剂量曲线应专门为个人群体.首先，在6个250毫升的玻璃瓶中浸泡100个种子，每个种子具有感兴趣的基因型，其中含有50毫升蒸馏水。在室温下以 100 RPM 摇动 8 小时，进行振动。然后，在烟气罩中，通过混合 EMS 液体和蒸馏水，准备 50 毫升五种不同浓度的 EMS 溶液。燃烧后，从五个烧瓶中抽取水。在含有吸收种子的五个烧瓶中加入50毫升EMS溶液。在 75 RPM 和室温下摇动烧瓶 16 小时。现在，将 EMS 溶液倒入空废瓶中，将经过处理的种子倒入放在空废瓶上的奶酪布上，以单独收集每次处理。使用额外的水来帮助浇灌种子。此外，将几毫升 EMS 不活化溶液喷洒到受污染的烧瓶壁上。将用过的移液器尖端浸入溶液中 24 小时。通过添加一卷 EMS 停用解决方案来治疗 24 小时，使已使用的 EMS 解决方案处于非活动状态。使用扭结，将 EMS 处理的种子放在奶酪布内，并在自来水下洗涤两小时。洗涤后，将每个种子分别移植到含有盆栽土壤的根部调节器中。在20至25摄氏度的温度下生长植物，16小时光。移植15天后，检查植物并计算未发芽的种子数量。记录植物生存数据。如果存活率在40-60%以内，则使用经过修饰的浓度进行第二轮剂量优化，直到在诱变实验后达到40至60%的所需致命性，将M2植物的叶组织收集到96井组织收集盒中。组织是从自有M1植物中生长的M2植物。每个 M1 工厂显示一个 M2 工厂。根据制造商的建议，使用植物DNA提取试剂盒提取DNA，并采用DNA纯化系统。然后，将两个微升的DNA提取物加载到有16个插槽的LV板中。使用波长为 260 和 280 纳米的分光光度计量化 DNA。用无核酸酶水稀释DNA浓度至每微升25纳米。通过将四个 96 井块中每井的 DNA 组合到池板中，同时保持每个样品的行和柱标识，创建四次 200 微升的 DNA 池。接下来，准备一个PCR主混合管的基因特定的底转，通过添加到每个管PCR缓冲液，向前和反向底转，和DNA聚合根据手稿。将主混合到 96 孔 PCR 板的孔中。然后，添加池化的 DNA 模板。将 PCR 管加载到热循环器中，并使用接触型材在热循环器上运行 PCR 反应。为了在不匹配的DNA之间产生异质复用，在不同的程序中孵育热循环器中的PCR产品。然后，将2.5微升自制的Cel-1内核糖酶添加到每个杂化PCR产品中。在45摄氏度下孵育45分钟。之后，通过添加2.5微升0.5摩尔EDTA在pH8终止Cel-1反应。将每个 Cel-1 处理的产品的 30.5 微升与 5 微升染料混合到 3%的 agarose 凝胶上，以 100 伏特运行两个半小时。为了去组织突变池，通过运行两个PCR反应的单个M2DNA，第一反应包含2.5微升的M2DNA和2.5微升的野生类型DNA，第二反应只包含5微升的M2DNA，确定突变体的酶度。该协议显示了EMS小谷物作物的变异和突变的特性。剂量曲线指示三种不同小麦所需的 50% 存活率的最佳 EMS 剂量。M2种群中容易识别的表型的存在证实了小谷物种群中诱变的有效性。以下是M2 TILLING种群中的突变表型、大麦M2种群中的白化病突变体、大麦M2种群中的氯突变体、Aegilops tauschii M2种群中粉红色变色的变异突变体以及Triticum单体M2种群中的低烧结突变体。在阿加罗斯凝胶平台上使用Cel-1的突变识别显示，通过独特的切割带在12个突变池中识别出一个潜在的突变池。突变池的去卷积决定了突变的酶性，并帮助跟踪突变到单个样本。A4池具有异质突变，在方框4和方框4野生型DNA样本中均有裂开带表示。H5池包含同源突变，因为独特的切割带只存在于方框5 H5野生型DNA样本中。最重要的步骤是确定 EMS 的最佳浓度，以达到 40 到 60% 的致命率。一旦一个具有高突变频率的 TILLING 种群被开发出来，它可用于任何感兴趣的基因的功能表征。此外，种群可能是作物改良有用基因变异的来源。TILLING 已广泛应用于模型和作物的功能基因组研究。获得的突变范围可以是错位、敲除、静默，甚至错用形式。Ems 是一个突变。因此，在处理 EMS 时，请使用适当的个人防护设备。去污染剩余的溶液和用过的移液器提示。

Research

JoVE Journal

Developmental Biology

Development of Targeting Induced Local Lesions IN Genomes (TILLING) Populations in Small Grain Crops by Ethyl Methanesulfonate Mutagenesis

Kidney Regeneration in Adult Zebrafish by Gentamicin Induced Injury

肾再生的成年斑马鱼庆大霉素性损伤

The kidney is essential for fluid homeostasis, blood pressure regulation and filtration of waste from the body. The fundamental unit of kidney function is ...

科研

发育生物学

Characterization of Thymic Settling Progenitors in the Mouse Embryo Using In Vivo and In Vitro Assays

Characterization of Thymic Settling Progenitors in the Mouse Embryo Using In Vivo and In Vitro Assays

胸腺表征祖定居在小鼠胚胎使用<em&gt;在体内</em&gt;和<em&gt;在体外</em&gt;测定

Characterizing thymic settling progenitors is important to understand the pre-thymic stages of T cell development, essential to devise strategies for T ...

Analysis of Zebrafish Larvae Skeletal Muscle Integrity with Evans Blue Dye

斑马鱼幼虫骨骼肌诚信与伊文思蓝分析

The zebrafish model is an emerging system for the study of neuromuscular disorders.  In the study of neuromuscular diseases, the integrity of the muscle ...

Technique to Target Microinjection to the Developing Xenopus Kidney

Technique to Target Microinjection to the Developing Xenopus Kidney

技术目标显微注射到显影<em&gt;爪</em&gt;肾

The embryonic kidney of Xenopus&#160;laevis (frog), the pronephros, consists of a single nephron, and can be used as a model for kidney disease. Xenopus ...

Development of an Ethanol-induced Fibrotic Liver Model in Zebrafish to Study Progenitor Cell-mediated Hepatocyte Regeneration

乙醇引起的肝纤维化模型的斑马鱼发展研究祖细胞介导的​​肝细胞的再生

Sustained liver fibrosis with continuation of extracellular matrix (ECM) protein build-up results in the loss of cellular competency of the liver, leading ...

Dissection and Immunofluorescent Staining of Mushroom Body and Photoreceptor Neurons in Adult Drosophila melanogaster Brains

Dissection and Immunofluorescent Staining of Mushroom Body and Photoreceptor Neurons in Adult Drosophila melanogaster Brains

成人果蝇脑内蘑菇体和感光神经元的解剖和荧光染色

Nervous system development involves a sequential series of events that are coordinated by several signaling pathways and regulatory networks. Many of the ...

Quantitative Whole-mount Immunofluorescence Analysis of Cardiac Progenitor Populations in Mouse Embryos

小鼠胚胎心肌内祖群的定量全贴免疫荧光分析

The use of ever-advancing imaging techniques has contributed broadly to our increased understanding of embryonic development. Pre-implantation development ...

Application of RNAi and Heat-shock-induced Transcription Factor Expression to Reprogram Germ Cells to Neurons in C. elegans

Application of RNAi and Heat-shock-induced Transcription Factor Expression to Reprogram Germ Cells to Neurons in C. elegans

rna 干扰和热休克诱导转录因子表达在重编程生殖细胞到神经元中的应用C. 线虫

Studying the cell biological processes during converting the identities of specific cell types provides important insights into mechanism that maintain ...

Cell Aggregation Assays to Evaluate the Binding of the Drosophila Notch with Trans-Ligands and its Inhibition by Cis-Ligands

Cell Aggregation Assays to Evaluate the Binding of the Drosophila Notch with Trans-Ligands and its Inhibition by Cis-Ligands

细胞聚集法评价果蝇切口与反式配体的结合及其对顺式配体的抑制作用

Notch signaling is an evolutionarily conserved cell-cell communication system used broadly in animal development and adult maintenance. Interaction of the ...

Controlled Cortical Impact Model of Mouse Brain Injury with Therapeutic Transplantation of Human Induced Pluripotent Stem Cell-Derived Neural Cells

人类诱导多能干细胞衍生神经细胞治疗移植小鼠脑损伤控制皮质影响模型

Traumatic brain injury (TBI) is a leading cause of morbidity and mortality worldwide. Disease pathology due to TBI progresses from the primary mechanical ...