Mittal Deepika, Mishra Shruti, Prajapati Ramgopal, Vadassery Jyothilakshmi
National Institute of Plant Genome Research, Aruna Asaf Ali Marg, New Delhi 110067, India.
National Institute of Plant Genome Research, Aruna Asaf Ali Marg, New Delhi 110067, India;
J Vis Exp. 2020 Aug 1(162). doi: 10.3791/61259.
Forward genetic screens have been important tools in the unbiased identification of genetic components involved in several biological pathways. The basis of the screen is to generate a mutant population that can be screened with a phenotype of interest. EMS (ethyl methane sulfonate) is a commonly used alkylating agent for inducing random mutation in a classical forward genetic screen to identify multiple genes involved in any given process. Cytosolic calcium (Ca) elevation is a key early signaling pathway that is activated upon stress perception. However the identity of receptors, channels, pumps and transporters of Ca is still elusive in many study systems. Aequorin is a cellular calcium reporter protein isolated from Aequorea victoria and stably expressed in Arabidopsis. Exploiting this, we designed a forward genetic screen in which we EMS-mutagenized the aequorin transgenic. The seeds from the mutant plants were collected (M1) and screening for the phenotype of interest was carried out in the segregating (M2) population. Using a 96-well high-throughput Ca measurement protocol, several novel mutants can be identified that have a varying calcium response and are measured in real time. The mutants with the phenotype of interest are rescued and propagated till a homozygous mutant plant population is obtained. This protocol provides a method for forward genetic screens in Ca reporter background and identify novel Ca regulated targets.
正向遗传学筛选一直是无偏向性鉴定参与多种生物学途径的遗传成分的重要工具。该筛选的基础是生成一个可针对感兴趣的表型进行筛选的突变群体。EMS（甲基磺酸乙酯）是一种常用的烷化剂，用于在经典正向遗传学筛选中诱导随机突变，以鉴定参与任何给定过程的多个基因。胞质钙（Ca）升高是应激感知时激活的关键早期信号通路。然而，在许多研究系统中，Ca的受体、通道、泵和转运体的身份仍然难以捉摸。水母发光蛋白是一种从维多利亚多管水母中分离出来并在拟南芥中稳定表达的细胞钙报告蛋白。利用这一点，我们设计了一种正向遗传学筛选，其中我们用EMS诱变了水母发光蛋白转基因植株。收集突变植株的种子（M1），并在分离群体（M2）中针对感兴趣的表型进行筛选。使用96孔高通量Ca测量方案，可以鉴定出几个具有不同钙反应的新型突变体，并进行实时测量。具有感兴趣表型的突变体被挽救并繁殖，直到获得纯合突变植株群体。该方案提供了一种在钙报告基因背景下进行正向遗传学筛选并鉴定新型钙调节靶点的方法。
