SITE-DIRECTED RECOMBINATION IN THE GENOME OF TRANSGENIC TOBACCO
SITE-DIRECTED RECOMBINATION IN THE GENOME OF TRANSGENIC TOBACCO
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DOI：
10.1007/bf00264442
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发表时间：
1990-09-01
期刊：
影响因子：
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通讯作者：
RUSSELL, S
中科院分区：
文献类型：
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作者：
ODELL, J;CAIMI, P;RUSSELL, S
植物基因组响应于噬菌体P1衍生的loxP-Cre位点特异性重组系统。重组发生在稳定整合在烟草基因组中的loxP位点，这表明Cre重组酶蛋白能够进入细胞核并定位到一个34bp的DNA序列。通过获得卡那霉素抗性来监测切除重组事件，卡那霉素抗性的获得是由于多聚腺苷基化信号序列的丢失导致嵌合的新霉素磷酸转移酶II基因的中断。分子分析证实切除确实发生了。当整合了loxP结构的植株被稳定地用嵌合cre基因重新转化时，以及当引入loxP结构的植株与携带嵌合Cre基因的植株杂交时，重组就发生了。根据表型分析，在50%-100%的含有这两个系统元素的植物中可以检测到位点特异性重组。在再转化后的2~3周和杂交幼苗的第一片叶中检测到卡那霉素抗性。这一证明了loxP-Cre系统在植物中的有效性，为开发该系统以指导定点整合和基因表达调控等目的提供了基础。
The plant genome responds to the bacteriophage P1-derived loxP-Cre site-specific recombination system. Recombination took place at loxP sites stably integrated in the tobacco genome, indicating that the Cre recombinase protein, expressed by a chimeric gene also stably resident in the genome, was able to enter the nucleus and to locate a specific 34 bp DNA sequence. An excisional recombination event was monitored by the acquisition of kanamycin resistance, which resulted from the loss of a polyadenylation signal sequence that interrupted a chimeric neomycin phosphotransferase II gene. Molecular analysis confirmed that the excision had occurred. Recombination occurred when plants with the integrated loxP construction were stably re-transformed with a chimeric cre gene and when plants with the introduced loxP construction were cross-bred with those carrying the chimeric Cre gene. As assayed phenotypically, site-specific recombination could be detected in 50%-100% of the plants containing both elements of the system. Kanamycin resistance was detected at 2-3 weeks after re-transformation and in the first leaf of hybrid seedlings. This demonstration of the effectiveness of the loxP-Cre system in plants provides the basis for development of this system for such purposes as directing site-specific integration and regulation of gene expression.