农杆菌介导的铁皮石斛遗传转化体系研究  

Study on Agrobacterium-mediated transformation system of Dendrobium officinale

作　　者：崔波[1,2] 刘佳[2] 王洁琼[2] 蒋素华[1] 武振江[2] 叶永忠[2] 

机构地区：[1]郑州师范学院,河南郑州450044 [2]河南农业大学生命科学学院,河南郑州450002

出　　处：《河南农业大学学报》2015年第2期208-212,共5页Journal of Henan Agricultural University

基　　金：郑州市科技攻关项目(074SGYS33205-2);河南省重点科技攻关项目(092102110128)

摘　　要：以铁皮石斛原球茎为受体,研究了农杆菌介导的铁皮石斛遗传转化体系,对遗传转化中的草甘膦筛选压力,菌液浓度,乙酰丁香酮浓度,预处理方法,侵染时间,美罗培南浓度等转化影响因子进行了优化。结果表明,摁压处理下的原球茎在添加浓度为200μmol·L-1乙酰丁香酮的菌液中侵染30 min,菌液浓度OD600为1.0,共培养48 h后,转入添加质量浓度为50 mg·L-1美罗培南和质量浓度为2.0 mg·L-1的草甘膦筛选培养基中,转化效率最高。经GUS组织化学染色及PCR分析鉴定,初步证明ACC合成酶反义基因已整合到铁皮石斛的基因组中。Using the protocorms of Dendrobium officinale as recipient materials,the genetic transformation system of Dendrobium officinale with Agrobacterium-mediated method was studied,and such influencing factors as PPT＇s resistant pressure,infection concentration of bacteria,concentration of AS,treatment methods of protocorms,infection time and concentration of Mer of the system were also optimized. The results showed that the press-treated protocorms were infected successfully by using the screening medium which contained 50 mg · L^-1 Mer and 2. 0 mg · L^-1 PPT after being treated with Agrobacterium tumefaciens suspension（ OD600= 1. 0,added 200 μmol·L^-1 AS） for 30 min and cocultured for 48 h. GUS histochemical staining and PCR analysis of the regenerated plants confirmed that the ACC synthase antisense gene had been introduced into the Dendrobium officinale genome.

关 键 词：铁皮石斛 原球茎 ACC合成酶 遗传转化 转化体系优化 

分 类 号：S435.72[农业科学—农业昆虫与害虫防治]