Name: investigating teliospore germination using microrespiration analysis and microdissection
Uploaded: 2018-05-13T14:00:00
Description: Watch this Scientific Journal Video about Investigating Teliospore Germination Using Microrespiration Analysis and Microdissection at JoVE.com

我们要感谢保罗. 弗罗斯特医生使用他的 microrespirometer, 妮可瓦格纳和亚历克斯贝尔的技术援助。这项工作由 NSERC 赠款资助 B.J.S。

1. 玉米芯感染
<ol><li>种植玉米玉蜀黍(cv 金矮脚鸡), 直到棒子形成, 并开始丝绸 (约60天)。</li>
	<li>与以前描述的7使用标准协议的区域性兼容的单倍体U. 玉米菌株。</li>
	<li>使用以前描述的7标准协议感染玉米棒子。</li>
</ol>2. Teliospore 收获
<ol><li>蒸压设备 (Büchner 漏斗, Büchner 烧瓶, 搅拌器, 250 毫升离心瓶, 扁平铲子, 和水) 使用标准...

<ol>
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担子 biotrophic 植物病原体每年造成数以亿计的农作物损失。绝大多数这些病原体产生冬孢子, 是真菌散布和性繁殖不可分割的组成部分。了解冬孢子的发展和发芽对理解这些真菌造成的破坏性疾病的传播至关重要。为了识别关键控制点的分子变化, 我们设计了一种方法来确定生理移位的时间, 另一种是在萌发的不同阶段分离冬孢子。et 等。(未发表) 注意到 teliospore 萌发的五个阶段的光显微术 ...

黑穗病真菌 (Ustilaginales) 由1600多个物种组成, 其中包括玉米、大麦和小麦等重要谷物作物, 每年造成的农作物损失数以亿计.1。这些真菌的特点是冬孢子的生产, 其中有黑暗的色素细胞壁和分散剂。冬孢子在寄主植物间分散的压力下保护遗传物质的作用, 并且可以在休眠状态持续多年 2.因此, 冬孢子是疾病传播的一个重要组成部分。
为了研究 teliospore 生物学, 我们的实验室利用了黑穗病真菌黑穗病玉米(U 玉米), 这是该病 "玉米常见黑穗病" 的因果动因。成熟的U. 玉米冬孢子的特点是生长骤停, 细胞代谢减少, 细胞呼吸的水平低 3.在有利的环境条件下 (e. g., 特定糖的存在), U. 玉米冬孢子发芽和完全减数分裂, 产生 basidiospores, 可引发新一轮的感染。发芽的特点是增加的呼吸, 恢复代谢活动, 并通过可见的形态学阶段的发芽4的进展。
发芽的初始阶段包括增加的呼吸和新陈代谢功能, 但是, 没有形态学的变化迹象。最初测量的呼吸变化在U. 玉米是50年前进行的, 测量耗氧量 manometrically 与华宝瓶设备5。我们开发了一种新的, 简单的方法来研究在 teliospore 发芽过程中的呼吸的精确变化, 用克拉克型 microrespirometer 测量在发芽的时间过程中的耗氧量。我们以前使用这种方法来研究野生型U. 玉米单倍体细胞和突变体与缺陷线粒体6之间呼吸速率的变化, 并在这里修改了该协议来研究 teliospore 呼吸过程中的变化。萌发。这为准确确定呼吸变化的时间提供了一种方法, 以便我们能够在发芽开始后适当的时间针对冬孢子进行研究, 以调查早期的分子事件。一旦 promycelia 从 teliospore 出现, 就可以微观地观察萌发过程, 但异步性质抑制了在特定阶段对足够冬孢子的隔离, 以供调查。我们开发了一种类似于体外受精的显微切割技术, 用于在不同形态的萌发阶段物理地收集冬孢子。

<table border="0" cellpadding="0" cellspacing="0" style="width:707px;" width="707">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:437px;">Streptomycin Sulfate</td>
			<td style="width:75px;">BioShop</td>
			<td style="width:124px;">STP101</td>
			<td style="width:71px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Kanamycin Sulfate</td>
			<td>BioShop</td>
			<td>KAN201</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Potato Dextrose Broth</td>
			<td>BD Difco</td>
			<td>254920</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">1 L Waring Laboratory blender</td>
			<td>Waring</td>
			<td>7011S</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Cheesecloth</td>
			<td>VWR</td>
			<td>470150-438</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Nalgene Polypropylene Desiccator with Stopcock</td>
			<td>ThermoFisher Scientific</td>
			<td>5310-0250</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Unisense MicroRespiration system</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;">MicroRespiration Sensor (O2)</td>
			<td>Unisense</td>
			<td>OX10</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">MicroOptode Meter Amplifier</td>
			<td>Unisense</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">MR-Ch Small</td>
			<td>Unisense</td>
			<td>MR-Ch</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">SensorTrace Rate Software</td>
			<td>Unisense</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">MicroRespiration Rack</td>
			<td>Unisense</td>
			<td>MR2-Rack</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">MicroRespiration Stirrer</td>
			<td>Unisense</td>
			<td>MR2-Co</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Microdissection system</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Axio Vert.A1 Inverted Light Microscope</td>
			<td>Zeiss</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Coarse Manipulator</td>
			<td>Narishige</td>
			<td>MMN-1</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Three-axis Hanging Joystick Oil Hydraulic Micromanipulator</td>
			<td>Narishige</td>
			<td>MMO-202ND</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Pneumatic Microinjector</td>
			<td>Narishige</td>
			<td>IM-11-2</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">TransferTip (ES)</td>
			<td>Eppendorf</td>
			<td>5175107004</td>
			<td></td>
		</tr>
	</tbody>
</table>

investigating teliospore germination using microrespiration analysis and microdissection

黑穗病真菌是几种破坏性农业病害的致病因子。它们的特点是冬孢子的生产, 这是厚壁分散剂。冬孢子可以保持休眠数十年。休眠的特点是低代谢率, 暂停大分子生物合成和大大降低呼吸水平。在接收到所需的环境信号后, 冬孢子发芽产生单倍体细胞, 这会引发新一轮的感染。Teliospore 萌发的特点是恢复大分子生物合成, 增加呼吸和戏剧性的形态学变化。为了精确测量发芽初期细胞呼吸的变化, 我们开发了一种使用克拉克型呼吸的简单协议。发芽的后期阶段是由特定的形态学变化来区分的, 但发芽是异步的。我们开发了一种显微切割技术, 使我们能够收集冬孢子在不同的发芽阶段。

利用克拉克型 microrespirometer 测量 teliospore 休眠和萌发过程中呼吸变化的方法, 我们证实休眠冬孢子与发芽相比具有低呼吸水平 (约1075µmol/小时/毫克)冬孢子 (2614 µmol/小时/毫克;图 1A)。这表示休眠冬孢子和冬孢子诱导发芽的平均呼吸速率有2.4 倍的变化。此外, 我们还发现, 诱导发芽的冬孢子在吸氧量上有45分钟的延迟 (图1B)。这是?...

Watch this Scientific Journal Video about Investigating Teliospore Germination Using Microrespiration Analysis and Microdissection at JoVE.com

Investigating Teliospore Germination Using Microrespiration Analysis and Microdissection

黑穗病真菌引起许多破坏性的农业病害。它们被分散为休眠冬孢子, 在响应环境提示时发芽。我们概述了两种方法来研究发芽过程中的分子变化: 测量呼吸增加, 以检测代谢活化和评估变化的分子事件通过隔离冬孢子在不同的形态学阶段。

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从长期来看，高解析度激光共聚焦时间流逝成像<em&gt;拟南芥子叶</em&gt;表皮萌发过程中

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调查的传播和毒性朊病毒样蛋白使用后生动物模式生物<em&gt;温度。线虫</em

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从细胞亚群特异性RNA提取使用激光捕获显微切割加上快速的免疫标记

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用基团特异 rDNA 引物对水生无脊椎动物进行遗传肠道含量分析的实验室规程

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Laser capture microdissection (LCM) has allowed gene expression analysis of single cells and enriched cell populations in tissue sections. LCM is a great ...