关键词: 山茶花, DNA提取, RAPD, 反应体系

Abstract: Camellia japonica L., a kind of rare flowers in China, is abundant in variety and difficult to classify and identify systematically. In order to find a technique for the classification and identification of Camellia japonica L. in DNA level and improve its molecular biological research, the optimal condition of DNA extraction and RAPD (Randomly Amplified Polymorphic DNA) amplification system were specially studied. In the experiment, the genomic DNA obtained from leaves by the method of modified CTAB had a fine purity with OD₂₆₀/OD₂₃₀ value about 1.90～2.0 and OD₂₆₀/OD₂₈₀ value about 1.80～1.83, a high yield with 150～200 μg·g^-1, and a good integrity of fragments above 20 kb. For the RAPD amplifying conditions of Camellia japonica L., the perfect reaction system in a 20 μL volume was considered to be 100 ng template DNA, 1× Buffer, 1.2 mmol·L^-1 Mg²⁺, 125 μmol·L^-1 dNTP (each), 0.5 μmol·L^-1 primer and 1 U Taq DNA polymerases. The anneal temperature was about 37℃, being adjusted lightly according to the different arbitrary primers. Presuming the phenolic compounds were excessive in leaves of Camellia japonica L., which needs to increase the quantity of β-Mercaptoethanol (4%) and add 2% PVP (Polyvinylpyrrolidone) in the DNA extraction solution to inhibit the oxidation of the phenolic compounds ultimately. It was also recommended that DNA extraction without 10% CTAB could still obtain the DNA with high purity and yield, moreover, the method was easy and steady. To get the good identical results in repeating experiments of RAPD amplification, it also should be noticed that each condition and step must be kept as consistent as possible, and make sure each reagent of reaction comes from the same source with the same concentration.

Key words: Camellia japonica L., DNA extraction, RAPD, reaction system