Name: screening of tobacco genotypes for phytophthora nicotianae resistance
Uploaded: 2022-04-15T12:30:00
Description: Watch this Scientific Journal Video about Screening of Tobacco Genotypes for Phytophthora nicotianae Resistance at JoVE.com

本研究由国家自然科学基金（31571738）和中国农业科技创新计划（ASTIP-TRIC01）资助。

1. 材料
<ol><li>获得烟草品种。 注：本实验“Beinhart1000-1”（Beinhart 1000）（BH）和“Xiaohuangjin1025”（XHJ）来自中国烟草种质资源国家中期数据库。BH 具有耐药性，而 XHJ 对 尼古丁酸疟原虫 感染敏感16。中国农业科学院烟草研究所保存的 尼古丁酸猪对 偶种0的田间分离物在整个研究过程中用于所有接种。</li>
</ol>2. 烟草种植基因型...

<ol>
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多种耐药性来源已被用于提高栽培烟草中的烟酸对虾的耐药性。单个显性R基因Php和Phl分别从Nicotiana plumbaginifolia和Nicotiana longiflora侵入10。雪茄烟草品种Beinhart 1000对P. nicotianae13具有最高的定量抗性水平。多个区间图谱实验表明，至少有六个数量性状位点（QTL）可能有助于该系的抗性13，<sup class="xr...

P. nicotianae 对许多茄科作物具有破坏性。可引起烟草“黑柄”1、马铃薯叶面和块茎腐病2、番茄和甜椒冠腐病和根腐病3、枸杞领和根腐病4。 烟粉可 攻击烟草植物的所有部位，包括任何生长阶段的根部、茎和叶片5。该疾病最常见的症状是茎的黑色基底。根部最初可见为水浸泡，然后坏死，叶子显示大的圆形病变5。这种疾病可能对温室和田间的烟草植物造成毁灭性打击6。控制 尼古丁酸对虾 最实用、最经济的方法是使用抗性品种7。然而，需要有效的筛查方案来鉴定烟草种质收集中的 耐烟酸假单胞菌种质。
已经描述了各种鉴定方法来评估烟草中的 硝酸对虾 的耐药性7，8，9，10，11，12，13，14，15，16。一般而言，已使用三种主要方法鉴定 耐烟化原虫烟草基因型。第一种包括在含有 P. nicotianae 的培养皿上混合菌丝体和琼脂培养基。然后将菌丝体在室温下在黑暗中培养2周。将1L去离子水加入菌丝体中并匀浆30秒。接种物保持在冰上，直到需要。在植物的每一侧做两个孔（直径1厘米，深4-5厘米），并将10毫升的接种物倒入每个孔中。然后用周围的土壤填充孔洞，每天监测疾病发展2周8，10。
在第二种方法中，用病原体感染的牙签接种植物。对于这种方法，植物应在移植后约6周使用，并且最小高度应为30厘米。将高压灭菌的牙签放在含有 P. nicotianae 菌丝体的培养物的表面上。然后将培养皿在室温下在光照下储存7天。然后，使用定植的牙签接种植物。将牙签插入第四和第五节点之间的烟杆中。每天监测植物5天9，15。此方法不适用于小苗。由于接种物是病原体感染的牙签，因此无法精确控制接种强度。
最常用的方法涉及燕麦粒进行接种。在这种情况下，通过将500mL燕麦和300mL去离子水在121°C下高压灭菌1小时，每天一次，持续3天来制备燕麦谷物。然后，将燕麦粒加入病原体定植培养基中。用石蜡膜密封培养皿，并在25°C的光照下孵育7-12天。在盆栽土壤上形成四个独立的5厘米深的孔，距离每株植物4厘米，每个孔中放置一个病原体感染的燕麦粒。潜伏期根据第一个地上症状发生的时间确定7，11，12，13，14，15，16。该方法高效，适用于大规模电阻筛选。然而，这种方法的一个局限性是接种物是病原体感染的燕麦粒，因此接种强度无法精确控制。
然而，这里介绍的是一种更准确的方法，适用于生长室阻力评估。与其他方法相比，接种物为游动孢子悬浮液，因此接种强度可控且可调。由于本研究中的烟草植物是在没有土壤的情况下种植的，因此结果更容易观察到。此外，从土壤中取样植物根部总是会对根部造成损害，从而诱发一系列生理反应17。在这种方法中，由于植物在没有土壤的情况下种植，因此可以消除对根系损伤的干扰。综上所述，该方法对于分子机理研究和精密育种更为实用。使用该协议，通常在5天内获得数据，在单个实验中评估200多种植物。

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		<tr>
			<td>(NH4)2SO4</td>
			<td>Sinopharm</td>
			<td>10002917</td>
			<td>Analytical Reagent</td>
		</tr>
		<tr>
			<td>(NH4)6 Mo7O24&#8226;2 H2O</td>
			<td>Sinopharm</td>
			<td>XW131067681</td>
			<td>Analytical Reagent</td>
		</tr>
		<tr>
			<td>1.5 ml Safe-lock Microcentrifuge Tubes</td>
			<td>Eppendorf</td>
			<td>30120086</td>
			<td>Used for Sample Extarction</td>
		</tr>
		<tr>
			<td>2 ml Safe-lock Microcentrifuge Tubes</td>
			<td>Eppendorf</td>
			<td>30120094</td>
			<td>Used for Sample Extarction</td>
		</tr>
		<tr>
			<td>Agar</td>
			<td>MDBio, Inc</td>
			<td>9002-18-0</td>
			<td>Materials of Culture Medium</td>
		</tr>
		<tr>
			<td>Analytical Balance</td>
			<td>AOHAOSI</td>
			<td>AX2202ZH</td>
			<td>Equipment</td>
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			<td>Autoclave</td>
			<td>Yamatuo</td>
			<td>SQ510C</td>
			<td>Equipment</td>
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			<td>Autoclave</td>
			<td>YAMATUO</td>
			<td>SQ510C</td>
			<td>Equipment</td>
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		<tr>
			<td>Beaker</td>
			<td>Bio Best</td>
			<td>DHSB-2L</td>
			<td>Materials of Culture Medium</td>
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		<tr>
			<td>Biological Incubator</td>
			<td>JINGHONG</td>
			<td>SHP-250</td>
			<td>Equipment</td>
		</tr>
		<tr>
			<td>Ca(NO3)2&#8226;4 H2O</td>
			<td>Sinopharm</td>
			<td>80029062</td>
			<td>Analytical Reagent</td>
		</tr>
		<tr>
			<td>CaCl2</td>
			<td>Sinopharm</td>
			<td>10005817</td>
			<td>Analytical Reagent</td>
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		<tr>
			<td>CuSO4&#8226;5 H2O</td>
			<td>Sinopharm</td>
			<td>10008218</td>
			<td>Analytical Reagent</td>
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		<tr>
			<td>Electromagnetic Oven</td>
			<td>Bio Best</td>
			<td>DHDCL</td>
			<td>Equipment</td>
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		<tr>
			<td>FeSO4&#8226;7 H2O</td>
			<td>Sinopharm</td>
			<td>10002918</td>
			<td>Analytical Reagent</td>
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		<tr>
			<td>Filter Paper</td>
			<td>Bio Best</td>
			<td>DHLZ-9CM</td>
			<td>Material</td>
		</tr>
		<tr>
			<td>Fluorescence Ration PCR Instrument</td>
			<td>Roche</td>
			<td>LightCycler96</td>
			<td>Equipment</td>
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		<tr>
			<td>Gauze</td>
			<td>Bio Best</td>
			<td>17071202</td>
			<td>Materials of Culture Medium</td>
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		<tr>
			<td>H3BO3</td>
			<td>Phytotechnology</td>
			<td>B210-500G</td>
			<td>Analytical Reagent</td>
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			<td>Hemocytometer</td>
			<td>Solarbio</td>
			<td>17072801</td>
			<td>Material for disease-resistant&#160; identification</td>
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		<tr>
			<td>K2SO4</td>
			<td>Sinopharm</td>
			<td>10017918</td>
			<td>Analytical Reagent</td>
		</tr>
		<tr>
			<td>KNO3</td>
			<td>Sinopharm</td>
			<td>10017218</td>
			<td>Analytical Reagent</td>
		</tr>
		<tr>
			<td>KT Foam Sheet</td>
			<td>Bio Best</td>
			<td>DHKTB</td>
			<td>Material for Seedling</td>
		</tr>
		<tr>
			<td>Low Constant Incubator</td>
			<td>Jinghong</td>
			<td>SHP-250</td>
			<td>Equipment</td>
		</tr>
		<tr>
			<td>Measuring Cylinder</td>
			<td>Bio Best</td>
			<td>DHBLLT-1000ML</td>
			<td>Materials of Culture Medium</td>
		</tr>
		<tr>
			<td>MgSO4&#8226;7 H2O</td>
			<td>Sinopharm</td>
			<td>10013080</td>
			<td>Analytical Reagent</td>
		</tr>
		<tr>
			<td>Microscope</td>
			<td>ECHO</td>
			<td>RVL-100-G</td>
			<td>Equipment</td>
		</tr>
		<tr>
			<td>MnCl2&#8226;4 H2O</td>
			<td>Sinopharm</td>
			<td>G5468154</td>
			<td>Analytical Reagent</td>
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		<tr>
			<td>Na2-EDTA</td>
			<td>Sinopharm</td>
			<td>G21410-250</td>
			<td>Analytical Reagent</td>
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			<td>NaH2PO4&#8226;2 H2O</td>
			<td>Sinopharm</td>
			<td>20040717</td>
			<td>Analytical Reagent</td>
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			<td>NH4NO3</td>
			<td>Sinopharm</td>
			<td>B64586-100g</td>
			<td>Analytical Reagent</td>
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		<tr>
			<td>Oatmeal</td>
			<td>Bio Best</td>
			<td>DHYMP-1.5KG</td>
			<td>Materials of Culture Medium</td>
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		<tr>
			<td>Petri Dish</td>
			<td>Bio Best</td>
			<td>DHPYM-9CM</td>
			<td>Material for disease-resistant&#160; identification</td>
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		<tr>
			<td>Pipettor</td>
			<td>THERMO</td>
			<td>S1</td>
			<td>Equipment</td>
		</tr>
		<tr>
			<td>Potting</td>
			<td>Bio Best</td>
			<td>DHYCXHP-12CM</td>
			<td>Material for Seedling</td>
		</tr>
		<tr>
			<td>Potting Soil</td>
			<td>Bio Best</td>
			<td>DHYMJZ-50L</td>
			<td>Seedling Material</td>
		</tr>
		<tr>
			<td>Punch</td>
			<td>Bio Best</td>
			<td>DHDKW</td>
			<td>Material</td>
		</tr>
		<tr>
			<td>qRT-PCR Plate</td>
			<td>Monad</td>
			<td>MQ50401S</td>
			<td>qRT-PCR Plate</td>
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		<tr>
			<td>SYBR&#160;Green Premix&#160;Pro Taq&#160;HS qPCR Kit</td>
			<td>Accurate Biology</td>
			<td>AG11718</td>
			<td>PCR Reagent</td>
		</tr>
		<tr>
			<td>Toothpick</td>
			<td>Bio Best</td>
			<td>DHYQ-900</td>
			<td>Material</td>
		</tr>
		<tr>
			<td>Total RNA Kit II</td>
			<td>Omega</td>
			<td>R6934-01</td>
			<td>PCR Reagent</td>
		</tr>
		<tr>
			<td>TransScript&#174;&#160;II One-Step gDNA Removal and cDNA Synthesis SuperMix</td>
			<td>Transgen</td>
			<td>AH311-02</td>
			<td>PCR Reagent</td>
		</tr>
		<tr>
			<td>Trays</td>
			<td>Bio Best</td>
			<td>DHYMTP-90G</td>
			<td>Material for Seedling</td>
		</tr>
		<tr>
			<td>Vermiculite</td>
			<td>Bio Best</td>
			<td>DHZS</td>
			<td>Seedling Material</td>
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		<tr>
			<td>Water Purification System</td>
			<td>HEAL FORCE</td>
			<td>HSE68-2</td>
			<td>Equipment</td>
		</tr>
		<tr>
			<td>ZnSO4&#8226;7 H2O</td>
			<td>Sinopharm</td>
			<td>10024018</td>
			<td>Analytical Reagent</td>
		</tr>
	</tbody>
</table>

screening of tobacco genotypes for phytophthora nicotianae resistance

由 卵菌黑芋引起的黑柄对烟草具有破坏性，这种病原体对许多茄科作物具有高度致病性。 P. nicotianae 很好地适应高温;因此，由于全球变暖，对这种病原体的研究在全世界的农业中变得越来越重要。耐 尼古丁酸的烟草植物品种通常通过接种由 尼古丁烷定 植的燕麦粒和监测疾病症状来筛查。然而，很难量化接种强度，因为在这种情况下，准确的接种至关重要。本研究旨在开发一种有效且可靠的方法来评估烟草对 烟酸假单胞菌感染的抵抗力。该方法已成功用于鉴定抗性品种，并通过实时荧光定量PCR证实了接种效率。本研究提出的抗性评价方法对于精密育种和分子机理研究具有高效实用性。

使用本文介绍的方法，用 尼古丁酸 对抗性品种BH和易感品种XHJ的4周龄植物进行挑战。该实验设计了三个重复，每个重复每组有8株植物。两种烟草品种BH和XHJ的 尼古丁酸对 虾感染如图 2所示。在接种后3天，对于XHJ，茎病变覆盖了茎围的约一半，并且叶子的一半略微枯萎;在耐药品种BH中，未观察到任何症状。在接种后4天，XHJ发生叶枯萎和严重的茎病变，而这些?...

Watch this Scientific Journal Video about Screening of Tobacco Genotypes for Phytophthora nicotianae Resistance at JoVE.com

Screening of Tobacco Genotypes for Phytophthora nicotianae Resistance

在这里，提出了一种方案，用于有效和准确地筛选幼苗中 烟叶植物 抗性的烟草基因型。这是精密育种以及分子机理研究的实用方法。

烟草基因型对 烟碱性植物 的筛选

Black shank, caused by the oomycetes Phytophthora nicotianae, is destructive to tobacco, and this pathogen is highly pathogenic to many solanaceous crops. ...

在这里，提出了一种方案，用于有效和准确地筛选幼苗中烟叶植物抗性的烟草基因型。它适用于精密育种以及分子机理研究。材料。从中国烟草种质资源国家中期基因库获取烟草品种Beinhart1000-1，精选的Beinhart1000 BH和Xiaohuangjin1025 XHJ。BH 耐药，而 XHJ 易受烟门疫霉菌感染。种植烟草基因型用于烟叶植物抗性评价。将烟草种子与蛭石混合，并在灭菌的盆栽土壤上轻轻地播种。将花盆放入生长室中。在16小时的光照/8小时的黑暗照片下保持25摄氏度的恒定温度。准备带有托盘和泡沫板的水培设备。种子发芽后，从盆栽土壤中刺出幼苗。用无菌去离子水轻轻清洗根部，然后将其移植到水培装置中。将设备置于25摄氏度的气候室中，在6小时的光照/8小时黑暗的照片期内，持续24小时。事先准备霍格兰营养液。将幼苗移植到带有Hoagland营养液的水培装置中。将设备置于25摄氏度的气候室中，在16小时的光照/8小时的黑暗照片期下，持续两周。烟生植物游动孢子悬浮液的制备.燕麦片琼脂培养基的制备。将33克燕麦片称入空心器皿中，加入1， 000毫升无菌水。在电磁炉上煮沸。燕麦片变粘后，通过一块无菌纱布过滤液体。将液体倒入1， 000毫升量筒中，并用无菌水将体积调节至1， 000毫升。将液体倒入玻璃试剂瓶中，加入18克琼脂。摇匀并高压灭菌混合物燕麦片琼脂培养基在121摄氏度下15分钟。在室温下放置30分钟。将约20毫升无菌的燕麦琼脂培养基倒入每个培养皿中。将培养皿置于室温下，在菌丝体培养之前彻底冷却。菌丝体栽培。事先准备一厘米直径的冲头和牙签，方法是将它们在121摄氏度的高压灭菌器中高压灭菌15分钟。在Phytophthora nicotianae菌丝体琼脂培养物中打孔，制成圆形菌丝垫。挑选菌丝垫，将菌丝体面朝下放在燕麦片琼脂培养基上，并在黑暗中以25摄氏度孵育菌丝体14天。烟生植物游动孢子悬浮液的制备.向每培养菌丝体中加入0.1%硝酸钾溶液，每皿15毫升，然后在4摄氏度下培养20分钟以诱导孢子囊。将菜肴保持在25摄氏度下25分钟，以释放游动孢子。将游动孢子悬浮液收集到烧杯中，并通过显微镜和血细胞计数器测量游动孢子浓度。用无菌水将游动孢子浓度调节到每毫升10至第四个游动孢子的一倍。鉴别抗病烟草品种。从Hoagland营养液中取出幼苗，并将根部浸入20毫升的Phytophthora nicotianae zoospore悬浮液中，在90毫米的培养皿中，在25摄氏度下在黑暗中浸泡三个小时，从而接种它们。接种后，将烟草幼苗放入新的培养皿中，加入10毫升无菌水，浸入根部。用两张滤纸盖住根部，保持根部湿润。将盘子保持在25摄氏度的16小时光照/8小时黑暗照片期。两到三天后，观察疾病的严重程度。对于对照处理，将烟草幼苗直接放入培养皿中，加入10毫升无菌水，浸根并用两张滤纸覆盖根部。评估烟道疫霉菌感染。接种疫苗后 4 至 5 天评估疾病严重程度。根据中国国家标准，对单个植物病害严重程度进行评分，评分范围从0到9。0级意味着整个植物没有症状。1级是指茎秆病变小于茎围的1/3或叶枯萎的1/3。3级是指茎周长的1/3至1/2之间或1/3至1/2的叶子轻微枯萎的茎病变。5级是指茎病变大于茎围的1/2，但不能完全围绕周长或叶片枯萎1/2至2/3之间。7级是指整个茎周长周围的茎病变或大于2/3的叶子枯萎。9级意味着植物看起来已经死了。使用以下公式计算疾病指数。疾病严重程度分为六个等级。具有代表性的结果。使用本文中介绍的方法，用尼古酸疫霉菌对抗性品种BH和易感品种XHJ的4周龄植物进行了挑战。在接种后三天，在XHJ中，茎病变覆盖了茎围的约1/2，叶子的1/2略有枯萎。在耐药品种BH中，未观察到任何症状。接种后4 d，XHJ出现叶枯萎和严重茎病变，而接种后 BH.At 5 d未出现这些症状，记录并按照中国烟草病虫害分级和调查方法国家标准记录和计算单个植物病害严重程度。BH的平均疾病指数为6.48，根据标准显示耐药性R，XHJ的平均疾病指数为76.85，根据标准显示易感性为S。为了确认接种效率，通过实时荧光定量PCR对相对病原体生物量进行了定量。实时荧光定量 PCR 的结果证实了表型观察结果。采用游动孢子悬浮接种法和燕麦粒接种法对BH与XHJ杂交BC4 F2群体的5个后代，分别对两个中间抗性品种K326和Yunyan87进行了评价。对小幼苗进行游动孢子悬浮法，对成株进行燕麦粒接种法。BC4 F2群体5个后代的病害指数为16.49~77.60，燕麦粒法为10.33~83.08。两种感染方法之间的耐药性分类大多是相互一致的。使用两种中间电阻品种K326和Yunyan87，评估结果显示两种方法均具有抗性。这些数据说明了尽管在不同生长期对烟草进行了两种接种方法之间的相关性。这里描述的方案是评估烟草对P.nicotianae幼苗阶段感染的抵抗力的有效和可靠的方法。该方案适用于育种和分子机理研究，因为燕麦粒法适用于成株的大规模抗性筛选。这两种方法可以互补使用。感谢您的收看。

Research

JoVE Journal

Biology

Screening of Tobacco Genotypes for Phytophthora nicotianae Resistance

Choice and No-Choice Assays for Testing the Resistance of A. thaliana to Chewing Insects

测试拟南芥耐咀嚼昆虫的选择和非选择性检测

Larvae of the small white cabbage butterfly are a pest in agricultural settings. This caterpillar species feeds from plants in the cabbage family, which ...

科研

生物学

MISSION esiRNA for RNAi Screening in Mammalian Cells

在哺乳动物细胞的RNAi筛选使命esiRNA

RNA interference (RNAi) is a basic cellular mechanism for the control of gene expression. RNAi is induced by short double-stranded RNAs also known as ...

Bioassays for Monitoring Insecticide Resistance

生物测定对杀虫剂的抗药性监测

Pest resistance to pesticides is an increasing problem because pesticides are an integral part of high-yielding production agriculture. When few products ...

Characterizing Herbivore Resistance Mechanisms: Spittlebugs on Brachiaria spp. as an Example

Characterizing Herbivore Resistance Mechanisms: Spittlebugs on Brachiaria spp. as an Example

表征草食动物的耐药机制：Spittlebugs臂形 SPP。为例

Plants can resist herbivore damage through three broad mechanisms: antixenosis, antibiosis and tolerance1. Antixenosis is the degree to which the plant is ...

A Fluorescent Screening Assay for Identifying Modulators of GIRK Channels

一个荧光识别GIRK通道调制器的筛选试验

G protein-gated inward rectifier K+ (GIRK) channels function as cellular mediators of a wide range of hormones and neurotransmitters and are expressed in ...

RNA Secondary Structure Prediction Using High-throughput SHAPE

使用高通量形状的RNA二级结构预测

Understanding the function of RNA involved in biological processes requires a thorough knowledge of RNA structure. Toward this end, the methodology dubbed ...

Assessing Murine Resistance Artery Function Using Pressure Myography

评估抗小鼠动脉功能使用压力Myography

Pressure myograph systems are  exquisitely useful in the functional assessment of small arteries, pressurized  to a suitable transmural pressure. The  ...

High-throughput Screening for Chemical Modulators of Post-transcriptionally Regulated Genes

高通量筛选转录后调控基因的化学调节剂

Both transcriptional and post-transcriptional regulation have a profound impact on genes expression. However, commonly adopted cell-based screening assays ...

Imaging Spatial Reorganization of a MAPK Signaling Pathway Using the Tobacco Transient Expression System

成像使用烟草瞬时表达系统MAPK信号通路的空间重组

Visualization of dynamic signaling events in live cells has been a challenge. We expanded an established transient expression system, the biomolecular ...

Comparison of Tobacco Host Cell Protein Removal Methods by Blanching Intact Plants or by Heat Treatment of Extracts

通过热烫完整植株或提取物热处理烟草宿主细胞蛋白去除方法的比较

Plants not only provide food, feed and raw materials for humans, but have also been developed as an economical production system for biopharmaceutical ...

烟草基因型对 烟碱性植物 的筛选

烟草基因型对烟碱性植物的筛选