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通过农杆菌介导法将双价载体(含抗真菌病的几丁质酶基因Chi和β-1,3-葡聚糖酶基因Glu)导入高羊茅愈伤组织细胞内,建立了高羊茅的农杆菌转化体系,获得了抗真菌病的转基因高羊茅植株。在转化过程中,确定了卡那霉素(Kan)筛选剂的浓度以200～300mg/L为宜,联合运用羧苄青霉素(Carb)与头孢霉素(Cef)作为抗菌素能得到好的脱菌效果;农杆菌的几种共培养方式对转化频率的影响表明,共培养基上垫一层滤纸,愈伤周围滴加浸染液,恢复培养1周后用甘露醇和抗生素液洗涤,是一种好的转化方式。对转化植株进行PCR分析和分子杂交及禾谷镰刀菌挑战接种,发现转化植株有高阳性率及抗真菌能力。
The bivalent vector (Chi and β-1,3-glucanase gene Glu containing antifungal gene chitinase gene) was introduced into the cells of tall fescue callus by Agrobacterium mediated method, Mao Agrobacterium transformation system, obtained anti-fungal transgenic tall fescue plants. In the process of transformation, the concentration of Kan screening agent was determined to be 200 ~ 300mg / L, the combination of carbenicillin and cef was used as antibiotic to get better sterilization The effect of several co-culture methods of Agrobacterium on the transformation frequency indicated that it was a good filter paper on the co-culture medium, and the immersion liquid was dropped around the callus and washed with mannitol and antibiotic solution after one week culture recovery Conversion method. The transformed plants were subjected to PCR analysis, molecular hybridization and challenge challenge with Fusarium graminearum. The transformed plants were found to have high positive rate and antifungal ability.