一种省力和可重复的触摸力信号突变屏幕协议,用于研究阿拉伯植物示范植物的三角性发生
Summary
一个温和的触摸力装载机是由人的头发刷,机械臂和控制器。发刷由安装在机器上的机械臂驱动,并定期移动,对植物施加触摸力。机器驱动的头发触摸强度可与手动应用的触摸强度相媲美。
Abstract
植物对细胞内和细胞外机械刺激(或力信号)作出反应,并产生特殊的形态变化,称为血性发生。在过去的几十年中,已经发现并报告了一些信号成分,这些成分参与了干扰(例如,钙离子结合蛋白和jasmonic酸生物合成酶)。然而,在力信号或异位磷发生研究中研究相对缓慢,主要归因于两个原因:需要人工操纵的触摸感应的异质性发生和力强度误差与人们的手触摸相关。为了提高植物有机体上外力装载的效率,建立了自动触摸力装载机。这款机械臂驱动式毛刷触感可提供省力且易于重复的触摸力模拟、无限轮的触摸重复和可调节的触摸强度。这种头发触摸力装载机可用于触摸力信号突变体的大规模筛选和植物造影的鸡学研究。此外,触摸材料,如人的头发,可以替代其他天然材料,如动物的头发,丝线和棉纤维。机器上的自动移动臂可配备喷水喷嘴和空气鼓风机,分别模拟雨滴和风的自然力。通过结合手工棉签触摸,结合这种自动发触摸力装载机,我们研究了两种力信号突变体MAP KINASE KINASE 1 (MKK1) 和 MKK2 工厂的触摸响应.对触摸力加载的野生型植物和两种突变体的现象进行了统计评价。他们在触摸响应方面表现出显著差异。
Introduction
植物异质性是1973年由Jaffe,MJ所创建的术语。它是一种植物对流,但不同于众所周知的光动或重力引起的阳光或重力刺激2,3。它描述了与周期性机械刺激相关的表型变化,在早期4,5中,植物学家经常观察到这些改变。雨滴、风、植物、动物和人类接触,甚至动物叮咬,都被认为是不同类型的梅查诺刺激,触发植物4,5的力信号。植物异质性的特征包括螺栓的延迟、较短的茎、草本植物中较小的玫瑰花/叶大小,以及木本植物6、7、8的较厚茎。这与在米莫萨植物或其他对美茶敏感的藤蔓中经常发现的异体或地皮反应不同,在这些快速接触反应中更容易观察到1、9、10。另一方面,由于生长反应缓慢,因此相对难以观察到三角性发生。在连续的力载刺激后,通常观察三重磷发生。植物触摸反应的这种独特特性使得使用人手触摸刺激来分离抗触摸信号突变体的正向遗传屏幕变得困难。
为了阐明强力信号转导途径和分子机制,在过去6年进行了分子和细胞生物学实验。12,13,14.这些研究提出,植物力信号受体主要由机械敏感电通道(MSC)和系绳MSC复合物组成,由膜跨度蛋白11、14的多体复合体组成。,15.细胞质Ca2+瞬态尖峰在初始接触后几秒钟内生成。风、雨或重力刺激可能与下游钙传感器相互作用,将力信号转用于核事件14、16、17、18。除了分子和细胞研究外,用手动手指触摸植物的正向遗传屏幕发现,植物激素和继发代谢物与随后的接触诱导(TCH)基因表达有关。触摸力加载13,19 。例如,到目前为止,从基因研究中已经发现了aos和opr320突变体。然而,在血性发生研究中应用前向遗传学的主要问题仍然是定量触摸反应水平和接触大量基因突变所需的大量劳动。个别植物。耗时的问题也仍然存在于手触摸为基础的突变屏幕14,20。例如,要完成一轮触摸力刺激,一个人需要触摸单个植物30-60次(每秒一次)。为了有足够的植物进行统计表型分析,触摸力加载过程通常需要20-50个相同基因型的单个植物。这种触摸力加载机制意味着一个人需要重复执行600-3,000次触摸选择一个基因型。这种类型的触摸通常需要每天重复3至5轮,这相当于大约1,800-15,000指或棉拭子触摸每天每个基因型的植物。训练有素的人通常需要在一天的多轮重复中,在理想的范围内保持多重接触的强度和力量,以避免力量和力量的很大变化。众所周知,血性发生是一个可饱和和剂量依赖的过程6,21,触摸力/强度成为成功触发植物触摸反应的关键。
为了消除与人相关的触摸力载荷,并将机械应用保持在可接受的误差范围14内,我们设计了一个自动触摸力装载机,以取代手工操作的触摸。机器有4个移动臂,每个都配有一个人发刷。此版本名为模型 K1,以指定其人发触摸力加载功能。如果在一台机器下定量测量4个基因型的血性发生或触摸反应,可以测量每个基因型40-48个个体。每轮触摸重复(每株触摸不到 60 次)使用移动速度可调机械臂持续不到 5 分钟。因此,K1 型触摸机上的工厂可以机械地刺激一天多轮,或者具有恒定的触摸力负载或最初编程的不同强度级别。
阿拉伯植物有机体模型,因此被选为测试全自动头发触摸力装载机应用的目标植物物种。由于有几个大型种子库可用于检索突变体的各种种质和开花的大小,阿拉伯兰多普西斯非常适合安装在使用型号 K1 触摸机的生长架上可用的空间。
K1 型自动触摸机由三个主要部件组成:(1) 由两个皮带驱动的线性执行器组成的 H 形金属机架,(2) 配备发刷的机械金属臂,以及 (3) 控制器。对于定制的 K1 型触摸机,每个 X/Y 轴模块由一个皮带驱动导轨、两个滑动块(红色)和一个 57 步进电机(预安装和拆卸)组成(图 1A,B)。上部水平执行器允许机械臂左右水平移动,下部垂直皮带驱动的线性执行器允许机械臂垂直上下移动(图 1B,图 2A).垂直执行器上安装了四个可拆卸的机械臂(图1C,图2B)。四个人类发刷被绑定到四个机械臂上,分别(图1C,图2B)。图1C中标有下面用粗体字构建K1型触摸机的所有机械部件(另见材料表)。
Protocol
Representative Results
Discussion
三毛磷发生是一种复杂的植物生长反应对机械扰动,这涉及到细胞信号和植物激素的作用网络。它是植物在不良环境条件下生存的适应性进化的结果25、26。机械触摸,特别是人的手指触摸和手持棉签触摸,已被选定来研究这种形态变化在以前的三元遗传学研究14,20。这种简化的触摸力加载版本可触发工厂触摸响应…
Declarações
The authors have nothing to disclose.
Acknowledgements
这项研究得到以下拨款的支持:31370315、31570187、31870231(中国国家科学基金会)、16100318、661613、16101114、16103615、16103817、AoE/M-403/16(香港RGC)。作者感谢巨峰精密自动化技术有限公司(中国深圳)提供图1所示的几张原理图。
作者们还要感谢张国宝和李华华为触摸力装载机的发展所做出的贡献。
Materials
| 4 hair brushes | customized | ||
| 4 robot arms with one holder | customized | 1000 mm length holder and 560 mm length robot arm | |
| 57 stepper motor | 57HS22-A | ||
| All purpose potting soil | Plantmate, Hong Kong | ||
| Arabidopsis plant seeds | Arabidopsis Biological Resource Centers, Columbus, OH | For arabidopsis seed purchase | |
| BIO-MIX potting substratum | Jiffy Products International BV, the Netherlands | 1000682050 | Two soils were mixed together to grow Arabidopsis. The ratio of All purpos potting soil and BIO-MIX is 1:2 |
| IL 1700 research radiometer | International Light, Newburyport, MA | The light intensity of both full-wavelength and photosynthetic active radiation can be measured. | |
| ImageJ | https://imagej.nih.gov/ij/download.html | Free downloaded software | |
| Ju Feng Precision and Automation Technology Limited | Shenzhen, China | For belt-driven linear actuators and other mechanical modules purchase | |
| Junction plate of the slide block | To fix the Y guide-rail module or Y auxiliary girder onto backs of slide blocks | ||
| Junction plate of the X axis module | customized | To connect the X guide-rail module and X auxiliary girder | |
| Slide block | |||
| WDT4045 X axis guide-rail module | 843 mm, customized | Pre-installed with two slide blocks and one 57 stepper motor | |
| WDT4045 Y axis guide-rail module | 1038 mm, customized | Pre-installed with two slide blocks and one 57 stepper motor | |
| X axis auxiliary girder | 843 mm, customized | Pre-installed with two slide blocks | |
| Y axis auxiliary girder | 1038 mm, customized | Pre-installed with two slide blocks |
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