山茶花叶片DNA提取及RAPD反应体系的研究

山茶花（Camellia japonica L.）是我国特产的名贵花卉之一，由于其品种繁多，在系统分类和品种鉴定上存在一定的难度。本研究针对山茶花的DNA提取方法和RAPD扩增体系进行了摸索，旨在为山茶花在DNA水平的分类鉴定和分子生物学方面研究打下一定的基础。实验采用改进的CTAB法提取山茶花叶片DNA，得到的DNA纯度较高，OD₂₆₀/OD₂₃₀值为1.90～2.0，OD₂₆₀/OD₂₈₀值为1.80～1.83，得率也较高，一般在150～200 μg·g^-1左右，片段完整性较好，大于20 kb，符合分子遗传实验的要求；在山茶花RAPD扩增条件的研究中，发现20 μL反应体系中加入100 ng DNA、125 μmol·L^-1 dNTP(each)、1.2 μmol·L^-1 Mg²⁺、1×Buffer、0.5 μmol·L^-1引物、1 U Taq酶最为合适，退火温度一般设为37℃左右，扩增产物的电泳条带数多、清晰、明亮。

Leaves DNA Extraction and RAPD Amplification of Camellia japonica L.

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.