高羊茅抗真菌病基因转化的研究  

Studies on Genetic Transformation with Resistance to Fungi Disease of Tall Fescue

作　　者：马生健[1] 徐碧玉[2] 曾富华[1] 卢向阳湖南农业大学生物技术系 

机构地区：[1]湛江师范学院生物系 [2]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

出　　处：《园艺学报》2006年第6期1275-1280,共6页Acta Horticulturae Sinica

基　　金：广东省科技计划项目(2002A2070402)

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200～300mg/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。Lawn is playing a more and more important role in human＇s 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 by Agrobacterium tumefacien, and the suitable transformation protocol of Agrobacterium of tall fescue was estabhshed. 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 cocuhivation methods of Agrobacterium showed that, putting some filter paper on cocultivation medium, and dripping infection solution of Agrobacterium around the calli, and using mannitol and antibiotic solution to washing ealli 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 plants had higher positive frequency and resistance to fungi disease than the control respectively.

关 键 词：高羊茅 草坪草 农杆菌 转化 真菌 

分 类 号：S688.4[农业科学—观赏园艺]