开发抗生素标记缺失型匍匐翦股颖抗除草剂特性。

Development of antibiotic marker-free creeping bentgrass resistance against herbicides.

机构信息

Grassland and Forages Division, National Institute of Animal Science, Cheonan, South Korea.

出版信息

Acta Biochim Biophys Sin (Shanghai). 2011 Jan;43(1):13-8. doi: 10.1093/abbs/gmq106.

Abstract

Herbicide-resistant creeping bentgrass plants (Agrostis stolonifera L.) without antibiotic-resistant markers were produced by Agrobacterium-mediated transformation. Embryogenic callus tissues were infected with Agrobacterium tumefaciens EHA105, harboring the bar and the CP4-EPSPS genes for bialaphos and glyphosate resistance. Phosphinothricin-resistant calli and plants were selected. Soil-grown plants were obtained at 14-16 weeks after transformation. Genetic transformation of the selected, regenerated plants was validated by PCR. Southern blot analysis revealed that at least one copy of the transgene was integrated into the genome of the transgenic plants. Transgene expression was confirmed by Northern blot. CP4-EPSPS protein was detected by ELISA. Transgenic plants remained green and healthy when sprayed with Basta, containing 0.5% glufosinate ammonium or glyphosate. The optimized Agrobacterium-mediated transformation method resulted in an average of 9.4% transgenic plants. The results of the present study suggest that the optimized marker-free technique could be used as an effective and reliable method for routine transformation, which may facilitate the development of varieties of new antibiotic-free grass species.

摘要

通过农杆菌介导的转化，生产出了不含抗生素抗性标记的抗草甘膦匍匐翦股颖（Agrostis stolonifera L.）植物。将胚性愈伤组织与携带 bar 和 CP4-EPSPS 基因的根癌农杆菌 EHA105 共培养，以获得对草丁膦和草甘膦的抗性。筛选出膦丝菌素抗性愈伤组织和植株。转化后 14-16 周，获得了在土壤中生长的植株。通过 PCR 验证了所选再生植株的遗传转化。Southern blot 分析表明，至少有一个转基因拷贝已整合到转基因植物的基因组中。通过 Northern blot 确认了转基因的表达。通过 ELISA 检测到 CP4-EPSPS 蛋白。当用含有 0.5%草铵膦铵或草甘膦的 Basta 喷洒时，转基因植物保持绿色和健康。优化的农杆菌介导的转化方法平均得到了 9.4%的转基因植物。本研究的结果表明，优化的无标记技术可作为常规转化的有效可靠方法，这可能有助于开发新的无抗生素草种品种。