Tyurin Alexander A, Suhorukova Aleksandra V, Deineko Igor V, Pavlenko Olga S, Fridman Viktoriia A, Goldenkova-Pavlova Irina V
Laboratory of Functional Genomics, Timiryazev Institute of Plant Physiology, Russian Academy of Sciences, Moscow, Russia.
Plant Methods. 2021 Oct 9;17(1):102. doi: 10.1186/s13007-021-00801-x.
Since the beginning of the use of reporter proteins for expression analysis, a variety of approaches have been developed and proposed; both qualitative and quantitative. The lack of simple methods for direct observation of gene expression in living organisms makes it necessary to continue to propose new methods. In this work, we consider a method for the quantitative analysis of the expression of thermostable lichenase from Clostridium thermocellum used as a sensitive reporter protein.
In this study, we report the design a high throughput fluorometric method for quantification of thermostable lichenase C. thermocellum using Congo red and further experimental verification of its relevance and efficiency in assessment of the functional role of regulatory sequences in the plant cell.
The specific interaction between the dye Congo red and [Formula: see text]-D-glucans formed the background for designing a high-throughput fluorometric assay for quantification of C. thermocellum thermostable lichenase as a reporter protein for plants. This assay (i) makes it possible to precisely measure the amount of reporter protein in a plant sample; (ii) has shown a high sensitivity for quantification of thermostable lichenase; (iii) is more time- and cost-efficient as compared with the Somogyi-Nelson assay; and (iv) is to the least degree dependent on the presence of the tested buffer components as compared with the Somogyi-Nelson assay.
自从开始使用报告蛋白进行表达分析以来，已经开发并提出了多种方法，包括定性和定量方法。由于缺乏在活生物体中直接观察基因表达的简单方法，因此有必要继续提出新的方法。在这项工作中，我们考虑了一种用于定量分析来自热纤梭菌的耐热地衣酶表达的方法，该酶用作敏感的报告蛋白。
在本研究中，我们报告了一种使用刚果红定量热纤梭菌耐热地衣酶的高通量荧光测定法的设计，并进一步通过实验验证了其在评估植物细胞中调控序列功能作用方面的相关性和效率。
染料刚果红与β-D-葡聚糖之间的特异性相互作用为设计用于定量热纤梭菌耐热地衣酶的高通量荧光测定法奠定了基础，该酶作为植物的报告蛋白。该测定法（i）能够精确测量植物样品中报告蛋白的量；（ii）对耐热地衣酶的定量显示出高灵敏度；（iii）与索莫吉-尼尔森测定法相比，更省时且成本效益更高；（iv）与索莫吉-尼尔森测定法相比，对测试缓冲液成分的依赖性最小。
