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Published on: November 23, 2011
[Establishment and optimization of sliver staining differential display of microtubers from Pinellia ternata in
Jian-Ping Xue1, Yue-Qin Huang, You-Ming Xu
1Anhui Key Laboratory of Plant Resources and Biology, School of Life Science, Huaibei Coal Industry Teachers' College, Huaibei 235000, China. xuejp2000@yahoo.com.cn
Objective:
In this study, orthogonal design was used to optinize 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, Mg2+, 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 20 microL DDRT-PCR system, it contained 10 x buffer, 150 micromol L(-1) dNTPs, 2 micromol L(-1) anchor primer, 1 micromol L(-1) arbitrary primer, 2.5 mmol L(-1) Mg2+, 0.6 U Taq DNA polymerase and 2.5 microg template cDNA. The effect of the five factors was in sequence of Taq DNA polymerase > template cDNA > dNTPs > Mg2+ > Primers. The optimum DDRT-PCR system will provide scientific reference basis for studying effecting character of P. ternata microtubers associated with genes expression.
