Novak Nicole G, Perez Frances G, Jones Richard W, Lawrence Susan D
Invasive Insect Biocontrol and Behavior Lab, United States Department of Agriculture (USDA), Agricultural Research Service (ARS).
Genetic Improvement for Fruits and Vegetables Lab, USDA-ARS.
J Vis Exp. 2019 May 15(147). doi: 10.3791/59153.
The multitrophic nature of gene expression studies of insect herbivory demands large numbers of biological replicates, creating the need for simpler, more streamlined herbivory protocols. Perturbations of chewing insects are usually studied in whole plant systems. While this whole organism strategy is popular, it is not necessary if similar observations can be replicated in a single detached leaf. The assumption is that basic elements required for signal transduction are present within the leaf itself. In the case of early events in signal transduction, cells need only to receive the signal from the perturbation and transmit that signal to neighboring cells which are assayed for gene expression. The proposed method simply changes the timing of the detachment. In whole plant experiments, larvae are confined to a single leaf which is eventually detached from the plant and assayed for gene expression. If the order of excision is reversed, from last in whole plant studies, to first in the detached study, the feeding experiment is simplified. Solanum tuberosum var. Kennebec is propagated by nodal transfer in a simple tissue culture medium and transferred to soil for further growth if desired. Leaves are excised from the parent plant and relocated to Petri dishes where the feeding assay is conducted with the larval stages of M. sexta. Damaged leaf tissue is assayed for the expression of relatively early events in signal transduction. Gene expression analysis identified infestation-specific Cys2-His2 (C2H2) transcription factors, confirming the success of using detached leaves in early response studies. The method is easier to perform than whole plant infestations and uses less space.
昆虫食草作用基因表达研究的多营养性质需要大量的生物学重复样本，因此需要更简单、更简化的食草实验方案。咀嚼式昆虫造成的扰动通常在整株植物系统中进行研究。虽然这种针对整个生物体的策略很常见，但如果在一片离体叶片上能够重复进行类似的观察，那么就没有必要采用这种方法。其假设是信号转导所需的基本元件存在于叶片本身。对于信号转导的早期事件而言，细胞只需要接收来自扰动的信号，并将该信号传递给用于基因表达检测的相邻细胞。所提出的方法只是改变了叶片离体的时间。在整株植物实验中，幼虫被限制在一片叶子上，这片叶子最终从植株上分离下来并进行基因表达检测。如果将切除顺序颠倒过来，从整株植物研究中的最后一步变为离体研究中的第一步，那么饲养实验就会得到简化。马铃薯品种Kennebec通过在简单的组织培养基中进行节段转移来繁殖，如果需要的话，可以转移到土壤中进一步生长。从母株上切下叶片，转移到培养皿中，在其中用烟草天蛾幼虫进行取食试验。对受损叶片组织进行信号转导相对早期事件的表达检测。基因表达分析鉴定出了侵染特异性的Cys2-His2（C2H2）转录因子，证实了在早期应答研究中使用离体叶片的成功。该方法比整株植物侵染实验更易于操作，且占用空间更小。
