欢迎访问林业科学,今天是

›› 2013, Vol. 49 ›› Issue (5): 121-127.doi: 10.11707/j.1001-7488.20130516

• 论文 • 上一篇    下一篇

杨树炭疽病菌原生质体遗传转化的建立及绿色荧光蛋白的表达

李思蒙1, 王永林1, 黄冬辉2, 田呈明1   

  1. 1. 北京林业大学林学院 省部共建森林培育与保护教育部重点实验室 北京 100083;2. 河北秦皇岛经济技术开发区园林绿化管理处 秦皇岛 066004
  • 收稿日期:2012-06-20 修回日期:2012-10-08 出版日期:2013-05-25 发布日期:2013-05-25
  • 通讯作者: 王永林

Establishment of a PEG-Mediated Genetic Transformation System and Expression of Green Fluorescence Protein in Colletotrichum gloeosporioides

Li Simeng1, Wang Yonglin1, Huang Donghui2, Tian Chengming1   

  1. 1. Key Laboratory for Silviculture and Conservation of Ministry of Education College of Forestry, Beijing Forestry University Beijing 100083;2. Landscaping Management Office of Qinhuangdao Economic and Technological Development Zone Qinhuangdao 066004
  • Received:2012-06-20 Revised:2012-10-08 Online:2013-05-25 Published:2013-05-25

摘要: 以杨树炭疽病菌菌株C1-5-2作为受体,通过PEG介导的原生质体转化法,将含有潮霉素B(hph)和GFP表达基因的质粒gGFP转入杨树炭疽病菌菌丝的原生质体中。幼嫩菌丝在0.7 mol·L-1 NaCl溶解的1% Lysing enzyme酶解液的作用下酶解210 min可以得到108·mL-1的原生质体; 在PEG介导下,通过含有潮霉素浓度为300 μg·mL-1的PDA选择培养基筛选转化子,每微克DNA获得41个转化子的平均转化效率。对转化子进行PCR鉴定表明: hph基因和GFP基因已经整合到杨树炭疽病菌转化子基因组中,通过荧光显微镜观察到转化子可以发出清晰的绿色荧光,且转化子的潮霉素抗性和GFP表达性状可以稳定遗传。

关键词: 杨树, 炭疽菌, 遗传转化, 原生质体, 绿色荧光蛋白

Abstract: We established a protoplasts transformation system mediated by polyethylene glycol (PEG), in which poplar anthracnose fungus Colletotrichum gloeosporioides strain C1-5-2 was used as the recipient, and obtained the transgenic transformants expressing a green fluorescence protein (GFP). Protoplasts were mixed with the plasmid gGFP containing the hygromycin phosphotransferase (hph) gene and GFP gene with PEG treatment. Fresh hyphae of recipient strain C1-5-2 were hydrolyzed in 20 mL enzyme solution containing 1% Lysing enzyme for 210 min, by which approximate 108 protoplasts·mL-1 were obtained. Transformation frequencies of 41 transformants per μg DNA were achieved after being screened on PDA medium containing hygromycin B concentration of 300 μg·mL-1. The verification with gDNA PCR amplifications indicated that the hph gene and GFP gene were indeed integrated into the genome of the C1-5-2 strain. Fluorescence observation showed clear and strong expression of the green fluorescent protein in fungal structures, and the GFP-tagged transformants were of genetic stability.

Key words: poplar, Colletotrichum, genetic transformation, protoplast, GFP

中图分类号: