天师栗叶片 DNA提取方法的建立

Establishment of DNAExtraction 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 , 严重影响后续的分子生物学操作 。本研究以树龄分别为 600、50、4 a 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 , 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 A. chinensis var . wilsonii , the quality of DNA ex- traction is often low, which seriously affects subsequent molecular biology operations . This study used mature leaves of 600, 50, and 4-year-old A. chinensis var . wilsonii plants as materials to establish a suitable method for extracting DNA from A. chinensis var . wilsonii (M1) and compared it with three standard plant DNA extraction methods (M2—M4) . The results showed that the concentration range for M1extraction of DNA ranged from 207. 19to 488. 98ng ● μL—1 , the av- erage value was 308. 42ng ● μL—1 ; the purity A260/A280 ratio ranged from 1. 90to 1. 98, with an average value of 1. 93. The A260/A230 ratio ranged from 1. 72to 1. 95, with an average value of 1. 86;DNA electrophoresis bands were clear , with mod- erate 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 M1is significantly superior to the other three meth- ods (M2—M4) in terms of concentration, purity, DNAbands , and IssR-PCR amplification products , making it suitable for the extraction of DNA from leaves of A. chinensis var . wilsonii.