Pramanik Dibyajyoti, Lee Keunsub, Wang Kan
Department of Agronomy, Iowa State University, Ames, IA, United States.
Crop Bioengineering Center, Iowa State University, Ames, IA, United States.
Front Plant Sci. 2024 Sep 18;15:1449409. doi: 10.3389/fpls.2024.1449409. eCollection 2024.
The reporter system has demonstrated great potential as a visible marker to monitor gene expression in both transiently and stably transformed plant tissues. Ectopic expression of the reporter leads to bright red pigmentation in plant tissues that do not naturally accumulate betalain. Unlike traditional visual markers such as β-glucuronidase (GUS), luciferase (LUC), and various fluorescent proteins, the reporter system does not require sample sacrifice or special equipment for visualizing the gene expression. However, a robust quantitative analysis method for betalain content has been lacking, limiting accurate comparative analyses. In this work, we present a simple and rapid protocol for quantitative evaluation of expression in transgenic plant tissues. Using this method, we demonstrate that differential expression can be quantified in transiently transformed leaf tissues, such as agroinfiltrated leaves, and in stable transgenic maize tissues, including seeds, leaves, and roots. We found that grinding fresh tissues with a hand grinder and plastic pestle, without the use of liquid nitrogen, is an effective method for rapid betalain extraction. Betalain contents estimated by spectrophotometric and High-Performance Liquid Chromatography (HPLC) analyses were highly consistent, validating that our rapid betalain extraction and quantification method is suitable for comparative analysis. In addition, betalain content was strongly correlated with expression level in agroinfiltrated leaves, suggesting that our method can be useful for monitoring transient transformation efficiency in plants. Using our rapid protocol, we quantified varying levels of betalain pigment in leaves, ranging from 110 to 1066 mg/kg of tissue, and in maize samples, ranging from 15.3 to 1028.7 mg/kg of tissue. This method is expected to streamline comparative studies in plants, providing valuable insights into the effectiveness of various promoters, enhancers, or other regulatory elements used in transgenic constructs.
报告系统已展现出巨大潜力，作为一种可见标记物可用于监测瞬时和稳定转化的植物组织中的基因表达。报告基因的异位表达会使植物组织中出现亮红色色素沉着，而这些组织通常不会自然积累甜菜红素。与传统的视觉标记物如β - 葡萄糖醛酸酶（GUS）、荧光素酶（LUC）以及各种荧光蛋白不同，该报告系统无需牺牲样品或使用特殊设备来观察基因表达。然而，一直缺乏一种用于甜菜红素含量的可靠定量分析方法，这限制了准确的比较分析。在这项工作中，我们提出了一种简单快速的方案，用于定量评估转基因植物组织中的表达。使用该方法，我们证明在瞬时转化的叶片组织（如农杆菌浸润的叶片）以及稳定转基因玉米组织（包括种子、叶片和根）中，可以对差异表达进行定量。我们发现，使用手动研磨机和塑料杵研磨新鲜组织，不使用液氮，是快速提取甜菜红素的有效方法。通过分光光度法和高效液相色谱（HPLC）分析估算的甜菜红素含量高度一致，验证了我们快速的甜菜红素提取和定量方法适用于比较分析。此外，甜菜红素含量与农杆菌浸润叶片中的表达水平密切相关，表明我们的方法可用于监测植物中的瞬时转化效率。使用我们的快速方案，我们定量了叶片中不同水平的甜菜红素色素，范围为每千克组织110至1066毫克，以及玉米样品中每千克组织15.3至1028.7毫克。该方法有望简化植物中的比较研究，为转基因构建体中使用的各种启动子、增强子或其他调控元件的有效性提供有价值的见解。
