摘要
为了构建限制性内切酶介导的整合(REMI)技术介导的红曲霉(Monascus anka)遗传转化系统,以潮霉素B作为抗性筛选标记,pBC-Hygro、pCB1003、pAN7-1作为转化质粒,采用REMI技术转化红曲霉。结果表明,用REMI技术介导红曲霉转化时,质粒pBC-Hygro和pCB1003适合于红曲霉的转化。环状质粒与线性质粒的转化率无显著差别;在用REMI技术介导转化时添加酶切缓冲液不利于转化;原生质体浓度为1×107～1×108个/mL时,每微克质粒可获得的潮霉素B抗性转化子为2800～3200个;HindⅢ介导转化时的最佳酶用量为105～120U;在质粒用量为8μg/100μL时转化率最高。
To establish a genetic transformation system of Monascus anka by restriction enzyme-mediated integration（REMI）,using the hph gene as selectable marker and pBC-Hygro,pCB1003 and pAN7-1 as vector,the fungus M.anka was transformed to be hygromycin B-resistant by REMI.Addition of restriction enzymes to transformation mixtures resulted in increase of transformation rate when using pBC-Hygro and pCB1003 as vectors.The transformation rate had no significant difference no matter if vectors were digested by restriction enzyme or not.Addition of enzyme digestion buffer resulted in reducing of transformation.Protoplasts at the concentration of 1×107～1×108 mL were fit for transforming M.anka,transformation number was 2 800～3 200 ind./μg vector DNA.The optimum Hind III and vector dose was 105～120 U and 8 μg/100 μL,respectively.
出处
《湖北农业科学》
北大核心
2012年第18期4129-4133,共5页
Hubei Agricultural Sciences
基金
贵州省科技厅农业攻关项目(黔科合NY字[2008]3058)
贵州大学研究生创新基金项目(校研农2010003)
