园艺学报 ›› 2006, Vol. 33 ›› Issue (6): 1275-1280.

• 研究论文 • 上一篇    下一篇


马生健1, 2;徐碧玉3;曾富华1;卢向阳2   

  1. (1 湛江师范学院生物系, 广东湛江524048; 2 湖南农业大学生物技术系, 湖南长沙410128; 3 中国热带农业科学院热带作物国家重点实验室, 海南海口571101)
  • 收稿日期:2006-04-10 修回日期:2006-04-14 出版日期:2006-12-25 发布日期:2006-12-25

Studies on Genetic Transformation with Resistance to Fungi Disease of TallFescue

Ma Shengjian1, 2;Xu Biyu3;Zeng Fuhua1;Lu Xiangyang2   

  1. (1Department of Biology, Zhanjiang Normal College, Zhanjiang, Guangdong 524048, China; 2Department of Biotechnology, Hunan Agricultural University, Changsha, Hunan 410128, China; 3 The State Key Laboratory of Tropical Crops Biotechnology,Chinese Academy of Tropical Agricultural Sciences, Haikou, Hainan 571101, China)
  • Received:2006-04-10 Revised:2006-04-14 Online:2006-12-25 Published:2006-12-25

摘要: 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200~300 mg/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。

关键词: 高羊茅, 草坪草, 农杆菌, 转化, 真菌

Abstract: Lawn is playing a more and more important role in humanps life, but its fungi disease is a important problem which should be solved urgently in the maintenance of lawn. In the paper, the binary vector harboring Glu and Chi gene was transformed into calli of tall fescue mediated byAg robacterium tum efacien, and the suitable transformation protocol of Agrobacterium of tall fescue was established. In the transformation, we confirmed that, the optimum concentration of selective agent kanamycin was 200 - 300 mg/L, the combination of carbercillin and cefotaxine as inhibitive agents had good inhibitive effect to Agrobacterium; The results of several cocultivation methods of Agrobacterium showed that, putting some filter paper on cocultivation medium, and dripping infection solution of Ag robacterium around the calli, and usingmannitol and antibiotic solution to washing calli 1 week after resuming cultivation, was the best way of transformation. The results of molecular
detection of PCR and Southern-blotting and challenge inoculation experiment with F. graminearum Schwabe showed that, transformed p lants had higher positive frequency and resistance to fungi disease than the control respectively.

Key words: Tall fescue, Turfgrass, Agrobacterium tumefacien, Transformation, Fungi