Intein-mediated Cre protein assembly for transgene excision in hybrid progeny of transgenic Arabidopsis
Intein-mediated Cre protein assembly for transgene excision in hybrid progeny of transgenic Arabidopsis
复制标题
内含肽介导的 Cre 蛋白组装用于转基因拟南芥杂交后代中的转基因切除
DOI：
10.1007/s00299-016-2015-x
复制
发表时间：
2016-10-01
影响因子：
6.2
通讯作者：
Luo, Keming
中科院分区：
文献类型：
--
作者：
Ge, Jia;Wang, Lijun;Luo, Keming
开发了一种恢复互补分裂Cre重组活性的方法，以切除转基因作物杂交后代中的转基因。转基因作物的生物安全性日益受到关注，目前已成为影响公众接受转基因作物的一个限制因素。已经开发了几种方法来生产无选择标记基因的转基因作物。然而，据报告，没有广泛适用于杂交作物的战略。先前的研究已经证明互补分裂Cre重组酶在转基因植物中恢复重组活性。在本研究中，我们发现当Cre重组酶在Asp232/Asp233（866 bp）处裂解时，由分裂内含肽Synechocystis sp. DnaE介导的分裂Cre重组酶具有较高的重组效率。此外，我们还构建了两个分别含有NCre 866-In和Ic-CCre 866片段的植物表达载体pCA-NCre-In和pCA-Ic-CCre。转化后，产生具有一个单拷贝的转基因拟南芥的亲本系并用于杂交。GUS染色结果表明，split-Cre重组活性片段通过杂交在转基因植株的杂交后代中得以重组，并有效地切除了两侧带有两个loxP位点的外源基因。我们的策略可能提供一个有效的方法来产生下一代的转基因杂交作物没有生物安全的问题。
An approach for restoring recombination activity of complementation split-Cre was developed to excise the transgene in hybrid progeny of GM crops. Growing concerns about the biosafety of genetically modified (GM) crops has currently become a limited factor affecting the public acceptance. Several approaches have been developed to generate selectable-marker-gene-free GM crops. However, no strategy was reported to be broadly applicable to hybrid crops. Previous studies have demonstrated that complementation split-Cre recombinase restored recombination activity in transgenic plants. In this study, we found that split-Cre mediated by split-intein Synechocystis sp. DnaE had high recombination efficiency when Cre recombinase was split at Asp232/Asp233 (866 bp). Furthermore, we constructed two plant expression vectors, pCA-NCre-In and pCA-Ic-CCre, containing NCre866-In and Ic-CCre866 fragments, respectively. After transformation, parent lines of transgenic Arabidopsis with one single copy were generated and used for hybridization. The results of GUS staining demonstrated that the recombination activity of split-Cre could be reassembled in these hybrid progeny of transgenic plants through hybridization and the foreign genes flanked by two loxP sites were efficiently excised. Our strategy may provide an effective approach for generating the next generation of GM hybrid crops without biosafety concerns.