丹参叶柄遗传转化体系的建立及EDT1基因的导入

Establishment of genetic transformation system of Salvia miltiorrhiza with oetiole as explants and introduction of EDT1 gene

【目的】为了建立和完善以丹参叶柄为外植体的遗传转化体系.【方法】以丹参品系HLM为受体,以携带EDT1基因的表达载体pCB3000-EDT1为目的基因供体,采用农杆菌介导法进行遗传转化,对转化的关键因子进行分析.【结果】最佳的转化体系条件为以14 d叶龄叶柄为外植体,农杆菌液浓度D_(600)=0.4浸染15 min,以MS+2.0 mg/L 6-BA+1.0 mg/L NAA愈伤诱导培养基.共获得44个克隆的142个阳性再生株.经PCR检测和干旱胁迫鉴定,获得抗旱性显著提高的再生株系4个.【结论】初步建立了以丹参叶柄作为外植体的遗传转化体系,为丹参遗传转化体系的研究和应用奠定了基础.

【Objective】To establish and improve the genetic transformation system of Salvia miltiorrhiza with the petiole as the explant.【Method】S.miltiorrhiza(line HLM)was used as the receptor,and the synthetic vector pCB3000 carrying EDT1 gene was used as the target gene donor to analyze the key factors of agrobacterium-mediated transformation.【Result】The optimum transformation conditions were as follows:14 days petiole as explant;concentration of Agrobacterium tumefaciens liquid D_(600)=0.4;immersion for 15 min;MS+2.0 mg/L 6-BA+1.0 mg/L NAA as callus induction medium.142 positive regenerated strains from 44 clones were obtained.After PCR detection and drought stress identification,four regenerated strains with significantly improved drought resistance were obtained.【Conclusion】The genetic transformation system of S.miltiorrhiza using petiole as explants was established,which provides a foundation for the research and application of S.miltiorrhiza genetic transformation system.