Zhang Xia'nan, Cui Guanghong, Jiang Xihong, Huang Luqi
Institute of Chinese Materia Medica, Academy of Chinese Medical Sciences, Beijing 100700, China.
Zhongguo Zhong Yao Za Zhi. 2012 Aug;37(15):2257-61.
To establish a culture system for transgenic Salvia miltiorrhiza hairy roots.
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 x L(-1) Cef water for 5 min, inoculated on MS solid medium supplied with 400 mg x mL(-1) cef and 2.5 g x L(-1) Hyg, and then transfered to the 67-V liquid medium with 2.5 g x 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.
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.
Successfully in vitro culture system has been established in transgenic S. miltiorrhiza, and this research can lay foundations for the further genetic engineering applications.
建立转基因丹参毛状根的培养体系。
研究不同外植体、不同感染时间和不同共培养时间对丹参毛状根诱导的成功率。共培养后的外植体用400 g·L⁻¹头孢噻肟钠水溶液灭菌5分钟，接种于添加400 mg·mL⁻¹头孢噻肟钠和2.5 g·L⁻¹潮霉素的MS固体培养基上，完全灭菌后转接至含有2.5 g·L⁻¹潮霉素的67-V液体培养基中。进行绿色荧光蛋白（GFP）荧光检测以筛选阳性毛状根株系。采用聚合酶链反应（PCR）方法检测毛状根特异基因rolC。测定不同生长时期根系的生物量。采用高效液相色谱法（HPLC）测定转基因毛状根中二氢丹参酮I的含量。
丹参叶基部是诱导毛状根的理想外植体，成功率可达93.3%。农杆菌感染10分钟诱导效率可达63.3%。共培养2 - 3天诱导效果最佳。采用PCR方法结合GFP荧光检测鉴定阳性转化体可信度高。转基因毛状根生物量增加与次生代谢产物积累密切相关。
成功建立了转基因丹参的体外培养体系，该研究可为进一步的基因工程应用奠定基础。
