Renilla luciferase-based quantitation of Potato virus A infection initiated with Agrobacterium infiltration of N. benthamiana leaves
Renilla luciferase-based quantitation of Potato virus A infection initiated with Agrobacterium infiltration of N. benthamiana leaves
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DOI：
10.1016/j.jviromet.2009.12.006
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发表时间：
2010-03-01
影响因子：
3.1
通讯作者：
Makinen, K.
中科院分区：
文献类型：
--
作者：
Eskelin, K.;Suntio, T.;Makinen, K.
开发并优化了一种基于海肾荧光素酶 (Rluc) 活性灵敏检测的马铃薯病毒 A（PVA；马铃薯病毒科）基因表达的定量方法。该系统基于农杆菌渗透引发的感染以及随后检测 PVA::Rluc RNA 的翻译，该翻译通过病毒复制而增强，首先在最初感染的细胞内，然后在病毒传播后在新细胞内翻译和复制。萤火虫荧光素酶 (Fluc) 用作内部对照，以标准化 Rluc 活性。从农杆菌渗透 (h.p.i.) 后 48 小时开始，观察到运动缺陷突变体和复制缺陷突变体之间的 Rluc/Fluc 活性比有大约 10 倍的差异。来自野生型 (wt) PVA 的 Rluc 活性在 48 至 72 h.p.i 期间显着增加。 Rluc/Fluc活性与突变病毒明显不同。通过半定量 RT-PCR 对 Rluc 和 Fluc mRNA 进行定量表明海肾荧光素酶 (rluc) mRNA 水平的增加和减少与 Rluc 活性的变化一致。然而，mRNA 水平的微妙增加导致 Rluc 活性的显着变化。 PVA CP 积累通过酶联免疫吸附测定进行定量。 Rluc活性的增加与病毒积累密切相关。 (C) 2009 Elsevier B.V. 保留所有权利。
A quantitation method based on the sensitive detection of Renilla luciferase (Rluc) activity was developed and optimized for Potato virus A (PVA; genus Potyviridae) gene expression. This system is based on infections initiated by Agrobacterium infiltration and subsequent detection of the translation of PVA::Rluc RNA, which is enhanced by viral replication, first within the cells infected initially and later by translation and replication within new cells after spread of the virus. Firefly luciferase (Fluc) was used as an internal control to normalize the Rluc activity. An approximately 10-fold difference in the Rluc/Fluc activity ratio between a movement-deficient and a replication-deficient mutant was observed starting from 48 h post Agrobacterium infiltration (h.p.i.). The Rluc activity derived from wild type (wt) PVA increased significantly between 48 and 72 h.p.i. and the Rluc/Fluc activity deviated clearly from that of the mutant viruses. Quantitation of the Rluc and Fluc mRNAs by semi-quantitative RT-PCR indicated that increases and decreases in the Renilla reniformis luciferase (rluc) mRNA levels coincided with changes in Rluc activity. However, a subtle increase in the mRNA level led to pronounced changes in Rluc activity. PVA CP accumulation was quantitated by enzyme-linked immunosorbent assay. The increase in Rluc activity correlated closely with virus accumulation. (C) 2009 Elsevier B.V. All rights reserved.