烟草基因型对 烟碱性植物 的筛选
Summary
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。
Abstract
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。
Introduction
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。
Protocol
1. 材料 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。 2. 烟草种植基因型…
Representative Results
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?…
Discussion
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…
Disclosures
The authors have nothing to disclose.
Acknowledgements
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。
Materials
| (NH4)2SO4 | Sinopharm | 10002917 | Analytical Reagent |
| (NH4)6 Mo7O24•2 H2O | Sinopharm | XW131067681 | Analytical Reagent |
| 1.5 ml Safe-lock Microcentrifuge Tubes | Eppendorf | 30120086 | Used for Sample Extarction |
| 2 ml Safe-lock Microcentrifuge Tubes | Eppendorf | 30120094 | Used for Sample Extarction |
| Agar | MDBio, Inc | 9002-18-0 | Materials of Culture Medium |
| Analytical Balance | AOHAOSI | AX2202ZH | Equipment |
| Autoclave | Yamatuo | SQ510C | Equipment |
| Autoclave | YAMATUO | SQ510C | Equipment |
| Beaker | Bio Best | DHSB-2L | Materials of Culture Medium |
| Biological Incubator | JINGHONG | SHP-250 | Equipment |
| Ca(NO3)2•4 H2O | Sinopharm | 80029062 | Analytical Reagent |
| CaCl2 | Sinopharm | 10005817 | Analytical Reagent |
| CuSO4•5 H2O | Sinopharm | 10008218 | Analytical Reagent |
| Electromagnetic Oven | Bio Best | DHDCL | Equipment |
| FeSO4•7 H2O | Sinopharm | 10002918 | Analytical Reagent |
| Filter Paper | Bio Best | DHLZ-9CM | Material |
| Fluorescence Ration PCR Instrument | Roche | LightCycler96 | Equipment |
| Gauze | Bio Best | 17071202 | Materials of Culture Medium |
| H3BO3 | Phytotechnology | B210-500G | Analytical Reagent |
| Hemocytometer | Solarbio | 17072801 | Material for disease-resistant identification |
| K2SO4 | Sinopharm | 10017918 | Analytical Reagent |
| KNO3 | Sinopharm | 10017218 | Analytical Reagent |
| KT Foam Sheet | Bio Best | DHKTB | Material for Seedling |
| Low Constant Incubator | Jinghong | SHP-250 | Equipment |
| Measuring Cylinder | Bio Best | DHBLLT-1000ML | Materials of Culture Medium |
| MgSO4•7 H2O | Sinopharm | 10013080 | Analytical Reagent |
| Microscope | ECHO | RVL-100-G | Equipment |
| MnCl2•4 H2O | Sinopharm | G5468154 | Analytical Reagent |
| Na2-EDTA | Sinopharm | G21410-250 | Analytical Reagent |
| NaH2PO4•2 H2O | Sinopharm | 20040717 | Analytical Reagent |
| NH4NO3 | Sinopharm | B64586-100g | Analytical Reagent |
| Oatmeal | Bio Best | DHYMP-1.5KG | Materials of Culture Medium |
| Petri Dish | Bio Best | DHPYM-9CM | Material for disease-resistant identification |
| Pipettor | THERMO | S1 | Equipment |
| Potting | Bio Best | DHYCXHP-12CM | Material for Seedling |
| Potting Soil | Bio Best | DHYMJZ-50L | Seedling Material |
| Punch | Bio Best | DHDKW | Material |
| qRT-PCR Plate | Monad | MQ50401S | qRT-PCR Plate |
| SYBR Green Premix Pro Taq HS qPCR Kit | Accurate Biology | AG11718 | PCR Reagent |
| Toothpick | Bio Best | DHYQ-900 | Material |
| Total RNA Kit II | Omega | R6934-01 | PCR Reagent |
| TransScript® II One-Step gDNA Removal and cDNA Synthesis SuperMix | Transgen | AH311-02 | PCR Reagent |
| Trays | Bio Best | DHYMTP-90G | Material for Seedling |
| Vermiculite | Bio Best | DHZS | Seedling Material |
| Water Purification System | HEAL FORCE | HSE68-2 | Equipment |
| ZnSO4•7 H2O | Sinopharm | 10024018 | Analytical Reagent |
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Cite This Article
Liu, Y., Sun, M., Jiang, Z., Wang, X., Xiao, B., Yang, A., Meng, H., Cheng, L. Screening of Tobacco Genotypes for Phytophthora nicotianae Resistance. J. Vis. Exp. (182), e63054, doi:10.3791/63054 (2022).