Vavilov Institute of General Genetics, Russian Academy of Sciences, Gubkina str. 3, 119991 Moscow, Russia.
Anal Bioanal Chem. 2010 Jul;397(6):2289-93. doi: 10.1007/s00216-010-3770-0. Epub 2010 May 13.
Thermostable lichenase encoded by licB gene of Clostridium thermocellum can be used as a reporter protein in plant, bacterial, yeast, and mammalian cells. It has important advantages of high sensitivity and specificity in qualitative and quantitative assays. Deletion variants of LicB (e.g., LicBM3) retain its enzymatic activity and thermostability and can be expressed in translational fusion with target proteins without compromising with their properties. Fusion with the lichenase reporter is especially convenient for the heterologous expression of proteins whose analysis is difficult or compromised by host enzyme activities, as it is in case of fatty acid desaturases occurring in all groups of organisms. Recombinant desaturase-lichenase genes can be used for creating genetically modified (GM) plants with improved chill tolerance. Development of an analytical method for detection of fused desaturase-lichenase transgenes is necessary both for production of GM plants and for their certification. Here, we report a multiplex polymerase chain reaction method for detection of desA and desC desaturase genes of cyanobacteria Synechocystis sp. PCC6803 and Synechococcus vulcanus, respectively, fused to licBM3 reporter in GM plants.
热稳定木聚糖酶由热纤维梭菌的 licB 基因编码，可用作植物、细菌、酵母和哺乳动物细胞中的报告蛋白。它在定性和定量测定中具有高灵敏度和特异性的重要优点。LicB 的缺失变体（例如 LicBM3）保留其酶活性和热稳定性，并且可以在不损害其性质的情况下与靶蛋白进行翻译融合表达。与木聚糖酶报告蛋白融合对于分析困难或受宿主酶活性影响的蛋白质的异源表达特别方便，就像所有生物体中存在的脂肪酸去饱和酶的情况一样。重组去饱和酶-木聚糖酶基因可用于创建具有改善耐寒性的转基因 (GM) 植物。开发用于检测融合去饱和酶-木聚糖酶转基因的分析方法对于 GM 植物的生产及其认证都是必要的。在这里，我们报告了一种多重聚合酶链反应方法，用于检测 GM 植物中分别与 licBM3 报告蛋白融合的蓝藻集胞藻 PCC6803 和聚球藻 Vulcanus 的 desA 和 desC 去饱和酶基因。
