Key Laboratory of Resource Biology and Biotechnology in Western China (Ministry of Education), College of Life Science, Northwest University, Xi'an, 710069, China.
Physiol Plant. 2018 Nov;164(3):242-250. doi: 10.1111/ppl.12681. Epub 2018 Jun 19.
Forward genetic analysis, widely used to find new gene functions, benefits from the availability of mutants. At present, based on Agrobacterium-mediated plant transformation technology, many transfer (T)-DNA transformants have been created. However, cloning their T-DNA insertion sites, which enables identification of the mutated genes, is still challenging. In this study, we improved adapter ligation-mediated polymerase chain reaction (A-PCR), which mainly utilizes the Thermal Asymmetric interlaced reaction and Degenerate sequence-recognizing restriction Endonucleases (TADE). Using the new method TADE-mediated A-PCR (TADEA-PCR), we successfully cloned 22 of all the 24 junction sites in 10 Arabidopsis thaliana L. transformants that contained 12 T-DNA insertions in total, giving a success rate of 91.7%. In most cases, the two junction sites resulting from a single T-DNA insertion were simultaneously cloned. In addition, TADEA-PCR was able to clone more than two junction sites present in one transformant containing several T-DNA insertions. Overall, TADEA-PCR is a powerful technique for cloning T-DNA insertion sites.
正向遗传学分析广泛用于寻找新的基因功能，得益于突变体的可用性。目前，基于农杆菌介导的植物转化技术，已经创建了许多转移（T）-DNA 转化体。然而，克隆其 T-DNA 插入位点，从而鉴定突变基因，仍然具有挑战性。在这项研究中，我们改进了衔接子连接介导的聚合酶链反应（A-PCR），该方法主要利用热不对称交错反应和简并序列识别限制内切酶（TADE）。使用新方法 TADE 介导的 A-PCR（TADEA-PCR），我们成功克隆了包含 12 个 T-DNA 插入的 10 个拟南芥 L.转化体中的 24 个连接位点中的 22 个，成功率为 91.7%。在大多数情况下，单个 T-DNA 插入产生的两个连接位点同时被克隆。此外，TADEA-PCR 能够克隆一个含有多个 T-DNA 插入的转化体中存在的两个以上连接位点。总的来说，TADEA-PCR 是一种克隆 T-DNA 插入位点的强大技术。
