NAN fusions: a synthetic sialidase reporter gene as a sensitive and versatile partner for GUS
NAN fusions: a synthetic sialidase reporter gene as a sensitive and versatile partner for GUS
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DOI：
10.1046/j.1365-313x.2002.01422.x
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发表时间：
2002-11-01
期刊：
影响因子：
7.2
通讯作者：
Kavanagh, TA
中科院分区：
文献类型：
--
作者：
Kirby, J;Kavanagh, TA
GUS 仍然是许多基因融合应用的首选报告基因，因为所编码的酶具有无与伦比的灵敏度，并且可以轻松地在无细胞提取物中对其进行定量，并在细胞和组织中进行组织化学可视化。兼容且功能等效的报告基因将有助于同一植物中的双启动子研究和表达分析的内部标准化。为了寻找植物中未发现的候选酶活性（这可能构成新型 GUS 兼容报告系统的基础），我们研究了 nanH，这是一种产气荚膜梭菌基因，编码所谓的“小”细胞质唾液酸酶。然而，转基因植物中富含 AT 的天然 nanH 基因的表达并未导致可检测到的唾液酸酶活性。因此，合成了密码子优化的衍生物 NAN，其 GC 含量与高表达植物基因中的 GC 含量相似。 NAN 酶活性在稳定和瞬时转化的细胞中均高水平表达，具有与 GUS 相似的动力学和稳定性特性，并在 GUS 缓冲液中显示出最佳活性。此外，使用相应的基于甲基伞形酮的底物，可以在聚丙烯酰胺凝胶中同时可视化 NAN 和 GUS 活性，并使用组织化学底物 5-溴-4-氯-3-吲哚基 α-D-N-乙酰神经氨酸 (X-NeuNAc) 和 5-溴-6-氯-3-吲哚基 β-D-葡萄糖醛酸 (X-GlucM)，在整个幼苗和组织切片中同时可视化 NAN 和 GUS 活性，分别。
GUS continues to be the reporter of choice for many gene fusion applications, due to the unparalleled sensitivity of the encoded enzyme and the ease with which it can be quantified in cell-free extracts and visualized histochemically in cells and tissues. A compatible and functionally equivalent reporter gene would facilitate dual promoter studies and internal standardization of expression analyses in the same plant. A search for a candidate enzyme activity not found in plants, which might form the basis of a novel GUS-compatible reporter system, led us to investigate nanH, a Clostridium perfringens gene which encodes the so-called 'small' cytoplasmic sialidase. Expression of the native, AT-rich nanH gene in transgenic plants did not, however, result in detectable sialidase activity. For this reason, a codon-optimized derivative, NAN, was synthesized which possesses a GC content similar to that found in highly expressed plant genes. NAN enzyme activity was expressed at high levels in both stably and transiently transformed cells, possessed kinetic and stability properties similar to those of GUS, and showed optimal activity in GUS buffer. Moreover, NAN and GUS activity could be visualized simultaneously in polyacrylamide gels using the corresponding methylumbelliferone-based substrates, and in whole seedlings and tissue sections using the histochemical substrates 5-bromo-4-chloro-3-indolyl alpha-D-N-acetylneuraminic acid (X-NeuNAc) and 5-bromo-6-chloro-3-indolyl beta-D-glucuronide (X-GlucM), respectively.