Wei ZhiGang

DNA extraction of birch leaves by improved CTAB method and optimization of its ISSR system

The basic method of DNA extraction (CTAB) was improved as the multi-times STE-CTAB extraction method and used to extract the DNA of birch leaved in this experiment. Results showed that the improved method is suitable not only for genomic DNA extraction of birch but also for that of other plants. The purity of genomic DNA extracted by the multi-times STE-CTAB extraction method is higher than that by one time STE-CTAB method, and it does not need the process of RNase. The factors of influencing ISSR system were explored based on the genomic DNA of birch extracted by the two methods. The optimal conditions for ISSR system were determined as follows: Mg2+ concentration is 1.5–3.0 mmol·L−1, dNTP concentration 0.10–0.25 mmol·L−1, the quantity of Taq polymerase 0.5–2.0 U, template DNA 30–100 ng, and the concentration of primer is 0.2–0.4 μmol·L−1, and the reaction program was as: initial denaturation for 5 min at 94°C, 30 cycles of denaturation for 30 s at 94°C, annealing for 30 s at 51 °C, extension for 30 s at 72°C, and a final 7 min extension at 72 °C.

Construction and analysis of a forward and reverse subtractive cDNA library from leaves and stem of Polygonum sibiricum Laxm.under salt stress

Using cDNAs prepared from the leaves and stems of Polygonum sibiricum Laxm. treated with NaHCO3 stress for 48 h as testers and cDNAs from unstressed P. sibiricum leaves and stems as drivers library, suppression subtractive hybridization (SSH) was employed to construct a cDNA subtracted library, which contained 2 282 valid sequences including 598 ESTs in the stems forward SSH library and 490 ESTs in the stem reverse SSH library, 627 ESTs in the leaf forward SSH library and 567 in the leaf reverse SSH library. According to the functional catalogue of MIPs and the comparison of the reverse and forward SSH libraries of the stem and leaf, the responses to NaHCO3 stress were different between leaf and stem, except for the same trend in cell rescue defense and transport facilitation. The trend in the metabolism, energy, photosynthesis, protein synthesis, transcription, and signal transduction was opposite. RT-PCR analysis demonstrated that the expression of 12 putative stress related genes in the NaHCO3-treated leaves and stems was different from that in the untreated leaves and stems. This indicated that different mechanisms might be responsible for reactions of leaf and stem in P. sibiricum. The results from this study are useful in understanding the molecular mechanism of saline-alkali tolerance in P. sibiricum.