丹参转基因毛状根离体培养体系的建立及分析

目的:建立起丹参的转基因毛状根诱导及离体培养体系。方法:考察了不同外植体、不同农杆菌侵染时间和共培养时间诱导丹参毛状根的效率,共培养外植体用400 g.L-1Cef水除菌5 min,接种在MS+400 g.L-1Cef+2.5 g.L-1Hyg的固体培养基上,完全除菌后转接入6,7-V+2.5 g.L-1Hyg液体培养基继代培养,GFP荧光检测阳性毛状根,PCR检测农杆菌特征基因rolC,并测定不同生长时期的毛状根干重和二氢丹参酮I的积累。结果:用丹参叶片基部诱导毛状根,成功率可达到93.3%;农杆菌侵染10 min诱导效率最高为63.3%;共培养2～3 d诱导效果最好;PCR结合GFP荧光检测的方法鉴定阳性转基因材料具有较高的可信性;丹参毛状根生物量变化与次生代谢物积累之间有着紧密联系。结论:成功建立起丹参转基因毛状根离体培养体系,为进一步的基因工程应用打下基础。

Establishment and analysis of in vitro culture system for transgenic Salvia miltiorrhiza hairy roots

Objective : To establish a culture system for transgenic Salvia miltiorrhiza hairy roots. Method : Investigated the success rate of different explants,different infection time and different co-culture time to induce hairy roots of S. miltiorrhiza. Co-cultured explants were sterilizated with 400 g·L^-1 Cef water for 5 min,inoculated on MS solid medium supplied with 400 mg·mL^-1 cef and 2.5 g·L^-1 Hyg, and then transfered to the 67-V liquid medium with 2.5 g·L^-1 Hyg after complete sterilization. GFP fluorescence detection was performed to detect positive hairy root lines. PCR method to detect rolC gene which is the specific gene of hairy root. Biomass was determinated in different growth periods of root lines. HPLC was conducted to measure the content of dihydrotanshinone I of transgenic hairy roots. Result : Leaf base of S. miltiorrhiza was used as a perfect explant to Induce hairy roots,the success rate can reach 93.3%. Inducing efficiency was up to 63.3% after Agrobacterium infection for 10 min. Co-culture for 2-3 d can reach the best induced effect. It is a high credibiliy to use PCR method combined with detection of GFP fluorescence to identified positive transformants. There is a close contact between biomass increases and secondary metabolite accumulation of transgenic hairy roots. Conclusion : Successfully in vitro culture system has been established in transgenic S. miltiorrhiza, and this research can lay foundations for the further genetic engineering applications.