Morono Yuki, Kitagawa Wataru, Kimura Nobutada, Noda Naohiro, Nakamura Kazunori, Kamagata Yoichi
Institute for Biological Resources and Functions, National Institute of Advanced Industrial Science and Technology, Ibaraki, Japan.
Appl Environ Microbiol. 2007 Aug;73(15):4915-21. doi: 10.1128/AEM.00068-07. Epub 2007 May 25.
A specific marking and detection technique is a fundamental requirement for the safer use of genetically modified (GM) organisms. Here we propose a simple and effective method for directly marking functional transgenes in GM organisms. For that purpose, we introduced nucleotide substitutions (NS), based on the degeneracy of codons as markers (NS markers), into the bphC (2,3-dihydroxybiphenyl dioxygenase) and tomA3 (toluene-ortho-monooxygenase) gene frames using a PCR-based method. No change was observed in the enzyme activity of translated proteins, and alignments with homologous genes showed the uniqueness of the NS markers. Furthermore, we constructed tomA3 variations harboring NS markers in different positions. Although the translational products were identical, the constructed variation genes could be distinguished through their marker patterns by multiplex PCR, showing that NS markers could serve as product-specific tags for identifying individual GM organisms. This direct method of marking the functional transgene provides a simple, low-risk, and robust marking method without causing the gene functions to deteriorate.
一种特定的标记和检测技术是安全使用转基因生物的基本要求。在此，我们提出了一种直接在转基因生物中标记功能性转基因的简单有效方法。为此，我们基于密码子简并性，采用基于聚合酶链式反应（PCR）的方法，将核苷酸替换（NS）作为标记（NS标记）引入到bphC（2,3-二羟基联苯双加氧酶）和tomA3（甲苯邻位单加氧酶）基因框架中。翻译后的蛋白质的酶活性未观察到变化，与同源基因的比对显示了NS标记的独特性。此外，我们构建了在不同位置带有NS标记的tomA3变体。尽管翻译产物相同，但通过多重PCR可以根据其标记模式区分构建的变体基因，这表明NS标记可以作为产品特异性标签来识别单个转基因生物。这种直接标记功能性转基因的方法提供了一种简单、低风险且稳健的标记方法，不会导致基因功能恶化。
