摘要
目的:建立半夏试管小块茎DDRT-PCR反应最佳体系,为进一步研究半夏试管小块茎相关基因表达提供技术参考。方法:以半夏试管苗的叶柄、试管小块茎为材料,利用正交试验设计,研究模板。Mg2+,dNTPs,引物和DNA聚合酶等因素,对DDRT-PCR反应体系的影响。结果与结论:20μL的反应体系中含有dNTPs 150μmol.L-1,Taq酶0.6 U,锚定引物2μmol.L-1,随机引物1μmol.L-1,Mg2+2.5 mmol.L-1,2μL 10×buffer和2.5μg的cDNA模板。各因素影响程度依次为Taq酶>cDNA模板>dNTPs>Mg2+>引物。
Objective： In this study,orthogonal design was used to optiMize DDRT-PCR amplification system on Pinellia ternata microtubers in vitro in five factors four levels respectively. Method： P. ternata stems and microtubers in vitro were selected as explants. The effects of five kinds of factors were studied by orthogonal design method including emplate cDNA, Mg^2＋ , dNTPs, primers and Taq DNA polymerase, and in order to establish the optimum DDRT-PCR system of P. ternata microtubers in vitro. Result and Conclusion： A satisfactory DDRT-PCR technique system for P. ternata microtubers in vitro with desirable repeatability and polymorphic bands was established. In a total volume of 20μL DDRT-PCR system, it contained 10×buffer, 150μmol·L^-1 dNTPs, 2μmol·L^-1 anchor primer, 1μmol·L^-1 arbitrary primer, 2. 5 mmol·L^-1 Mg^2＋ , 0. 6 U Taq DNA polymerase and 2. 5 p,g template cDNA. The effect of the five factors was in sequence of Taq DNA polymerase 〉 template cDNA 〉 dNTPs 〉 Mg^2＋〉 Primers. The optimum DDRT-PCR system will provide scientific reference basis for studying effecting character of P. ternata microtubers associated with genes expression.
出处
《中国中药杂志》
CAS
CSCD
北大核心
2008年第19期2170-2174,共5页
China Journal of Chinese Materia Medica
基金
国家农业成果转化资金重点项目(05EFN213400124)
淮北市重大专项(06085)
