天师栗叶片DNA提取方法的建立

Establishment of DNA extraction method from leaves of Aesculus chinensis var. Wilsonii.

摘要:

提取高质量的基因组DNA是开展分子生物学研究的重要前提之一。天师栗由于叶片含有较多的多糖、多酚类物质,常常导致DNA 的提取质量不高,严重影响了后续的分子生物学操作。本研究以树龄分别为600年、50年和4年的天师栗植株的成熟叶片为材料,建立了适合于天师栗叶片DNA 的提取方法(M1),并将其与3种常见植物DNA提取方法(M2～M4)进行了比较。结果表明:M1提取DNA的浓度范围介于207.19~488.98 ng·μL -1之间,平均值为308.42 ng·μL -1；纯度A260/A280比值介于1.90~1.98,平均值为1.93；A260/A230比值介于1.72~1.95,平均值为1.86；DNA电泳条带清晰,亮度适中,点样孔干净无污染；ISSR-PCR能扩增出条带清晰、稳定性好、多态性高的PCR产物。M1提取出的DNA在浓度、纯度、DNA条带、ISSR-PCR扩增产物方面均明显优于其它3种方法(M2～M4),适合用于天师栗叶片DNA 的提取。

Abstract:

Extracting high-quality genomic DNA is one of the essential prerequisites for conducting molecular biology research. Due to the high content of polysaccharides and polyphenols in the leaves of Aesculus chinensis var Wilsonii, the quality of DNA extraction is often low, which seriously affects subsequent molecular biology operations. This study used mature leaves of 600, 50, and 4-year-old Aesculus chinensis var Wilsonii plants as materials to establish a suitable method for extracting DNA from Aesculus chinensis var Wilsonii (M1) and compared it with three standard plant DNA extraction methods (M2-M4). The results showed that the concentration range for M1 extraction of DNA ranged from 207.19 to 488.98 ng· μL-1, the average value was 308.42 ng· μL-1; the purity A260/A280 ratio ranged from 1.90 to 1.98, with an average value of 1.93. The A260/A230 ratio ranged from 1.72 to 1.95, with an average value of 1.86; DNA electrophoresis bands were clear, with moderate brightness, and the sample holes were clean and pollution-free; ISSR-PCR can amplify PCR products with clear bands, good stability and high polymorphism. The DNA extracted by M1 is significantly superior to the other three methods (M2~M4) in terms of concentration, purity, DNA bands, and ISSR-PCR amplification products, making it suitable for the extraction of DNA from leaves of Aesculus chinensis var. Wilsonii.