丹参不同愈伤组织诱导及遗传转化体系优化

Callus inducing on different phytohormone media and Agrobacterium mediated genetic transformation system optimizing of Salvia miltiorrhiza

关键词:  丹参， 愈伤， 诱导， 遗传转化

Key words:  Salvia miltiorrhiza, callus, induction, genetic transformation

摘要:

为了获得满足不同目的组织培养材料和稳定高效的遗传转化体系，该研究以丹参叶片和茎段为外植体，采用含不同浓度的植物激素（植物生长物质）的Murashige Skoog（MS）培养基，探索诱导丹参产生不同愈伤的条件；采用正交法考察浸染时间、共培养时间、筛选压等对农杆菌介导的丹参遗传转化体系的影响，并根据出芽率及转化阳性率优化丹参遗传转化体系。结果表明：（1）能较快诱导丹参叶片产生愈伤的是MS+0.5 mg·L 1 6 BA+0.5 mg·L 1 2，4 D；诱导茎较快产生愈伤的是MS+0.1mg·L 1 NAA+0.5 mg·L 1 6 BA； 1 mg·L 1反式玉米素（ZR）可能有利于诱导产生含有丹参酮的愈伤组织；1.0 mg·L 1 2,4 D较易诱导丹参愈伤组织生根。（2）以卡那霉素为筛选剂时农杆菌GV3101介导的丹参遗传转化的条件为浸染5 min、共培养1 d、卡那霉素30 mg·L 1筛选,经PCR鉴定转基因阳性率为60%；而用10 mg·L 1链霉素筛选阳性率达70%。该研究结果确定了丹参不同愈伤组织诱导条件，明确了以卡那霉素为筛选剂时农杆菌GV3101介导的丹参遗传转化的条件，换用10 mg·L 1链霉素筛选时体系更加稳定、更易操作、更易重复。

Abstract:

With the decoding of its genome, Salvia miltiorrhiza, belonging to labiatae (Lamiaceae) sage (Salvia Linn), is becoming an important model medicinal plant and is being widely studied. The earliest record of S. miltiorrhiza can be found in more than two thousand years ago in the Sheng Nong’s Herbal Classic in which S. miltiorrhiza was listed as one of the top grade drugs. S. miltiorrhiza has a long history in treatment of many cardiovascular and cerebrovascular diseases. The active components of S. miltiorrhiza are divided into two categories: water soluble phenolic acids and lipid soluble tanshinones. As an important medicinal mode plant with long history, the biosynthetic pathways of S. miltiorrhiza active contents tanshinones and phenolic acids attract growing research interests. An appropriate tissue culture condition and a simple, stable and efficient genetic transformation system are very important in the research of the plant S. miltiorrhiza. Based on former studies, we investigated culture conditions to induce different S. miltiorrhiza calluses using different phytohormones on Murashige Skoog medium. And through a four factor three level orthogonal design, we investigated the effects of submerging time, co culture time, screening pressure and concentrate of acetosyringone. The results of callus induction were that: MS+0.5 mg·L 1 6 BA+0.5 mg·L 1 2,4 D induce callus faster than other explants, while MS+0.1 mg·L 1 NAA+0.5 mg·L 1 6 BA was suitable in inducing stems; 1 mg·L 1 trans zeatin could induce brawn callus maybe contain more tanshinone, 1.0 mg·L 1 2,4 D could induce more roots on callus. And the confirmed agrobacterium mediated genetic transformation parameters for agrobacterium tumefaciens GV3101 harboring nptII gene plasmid were: submerging 5 min, co culture 1 d and screening at 30 mg·L 1 kanamycin, the positive ratio of transgenic plant by PCR verification was 60%. When submerging 5 min, co culture 1 d and screening at 10 mg·L 1 streptomycin the transgenic plant positive rate was 70%. The later transgenic system seemed more stable and easier to be repeated in this trial, maybe due to the less toxic of streptomycin which indicated that streptomycin maybe another candidates for transgenic screening in S. miltiorrhiza. The determination of different callus inducing condition and the optimizing of genetic transformation system provide useful appliancation in further researches of S. miltiorrhiza.