Plant Gene Engineering Center, South China Botanical Garden, Chinese Academy of Sciences, Guangzhou, China.
University of Chinese Academy of Sciences, Beijing, China.
Plant Biotechnol J. 2017 Nov;15(11):1420-1428. doi: 10.1111/pbi.12726. Epub 2017 Apr 20.
We have previously described a recombinase-mediated gene stacking system in which the Cre recombinase is used to remove lox-site flanked DNA no longer needed after each round of Bxb1 integrase-mediated site-specific integration. The Cre recombinase can be conveniently introduced by hybridization with a cre-expressing plant. However, maintaining an efficient cre-expressing line over many generations can be a problem, as high production of this DNA-binding protein might interfere with normal chromosome activities. To counter this selection against high Cre activity, we considered a split-cre approach, in which Cre activity is reconstituted after separate parts of Cre are brought into the same genome by hybridization. To insure that the recombinase-mediated gene stacking system retains its freedom to operate, we tested for new locations to split Cre into complementing fragments. In this study, we describe testing four new locations for splitting the Cre recombinase for protein fragment complementation and show that the two fragments of Cre split between Lys244 and Asn245 can reconstitute activity that is comparable to that of wild-type Cre.
我们之前描述了一种重组酶介导的基因堆叠系统，其中 Cre 重组酶用于在每轮 Bxb1 整合酶介导的位点特异性整合后去除不再需要的lox 位点侧翼 DNA。Cre 重组酶可以通过与表达 cre 的植物杂交方便地引入。然而，在许多代中维持高效的 cre 表达系可能是一个问题，因为这种 DNA 结合蛋白的高产量可能会干扰正常的染色体活动。为了应对这种对高 Cre 活性的选择，我们考虑了一种分裂 Cre 的方法，其中通过杂交将 Cre 的两个分开的部分引入同一基因组中，从而重新构成 Cre 活性。为了确保重组酶介导的基因堆叠系统保持其自由操作的能力，我们测试了新的位置将 Cre 分裂成互补片段。在这项研究中，我们描述了测试 Cre 分裂的四个新位置用于蛋白质片段互补，并表明在 Lys244 和 Asn245 之间将 Cre 分裂成两个片段可以重新构成与野生型 Cre 相当的活性。
