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[目的]研究蕨类植物DNA提取的方法,为进一步的遗传多样性以及分类学的研究提供基础。[方法]通过改变试剂的用量以及操作方法对CTAB法提取DNA方法进行了改进。①取0.5～1.0 g新鲜叶片,加液氮充分研磨成粉末,置于1.5 ml离心管中,加2%CTAB600μl及10μl巯基乙醇;②65℃水浴15 min(期间取出振荡1次),冷却;③加500μl氯仿/异戊醇(24:1)上下颠倒数次至液相深绿色;④12 000 r/min离心3 min;⑤取上清液至-1.5 ml离心管,重复步骤③、④;⑥取上清液至-1.5 ml离心管中,管内预装1 ml 95%乙醇。室温静置2 h;⑦12 000 r/min离心3 min;⑧弃上清,用70%乙醇(约4400μl)洗涤沉淀,真空干燥器干燥。干燥后加低温保存;⑨使用时加100μl超纯水溶解。-25℃保存。提取出的DNA样品中加超纯水50μl,混匀后,用石英比色杯于DU(R)800分光光度计中测定紫外消光值,根据其在260nm和280 nm波长处的光吸收值计算DNA产率,根据A_(260)/A_(280)判断DNA样品的纯度。DNA样品的浓度(μg/μl)为:在260 nm的紫外消光值×核酸稀释倍数×50/1 000。纯DNA样品A_(260)/A_(280)紫外消光值应为1.8,A_(260)/A_(230)值应大于2.0。A_(260)/A_(280)值大于1.9时,表明有RNA污染,小于1.6时,表明样品中存在蛋白质或酚污染。A_(260)/A_(230)值小于2.0时表明溶液中有残存盐和小分子杂质,如核甘酸、氨基酸、酚等。分别对常规CTAB法和改进CTAB法提取出的蕨类植物DNA进行琼脂糖凝胶电泳检测,以验证提取出DNA的质量。[结果]改进的CTAB法提取的蕨类DNA,其主带(基因组DNA)更加明亮,且与其他杂带区分明显,表明DNA的降解较少,DNA得率及纯度都有了很大提升。[结论]改良的CTAB提取法能够提取出高质量的蕨类植物DNA。
[Objective] The research aimed to study the DNA extraction method of ferns and provide the basis for further study of genetic diversity and taxonomy. [Method] The method of extracting DNA by CTAB method was improved by changing the amount of reagent and operation method. ① Take 0.5 ~ 1.0 g fresh leaves, add liquid nitrogen and thoroughly ground into powder, placed in 1.5 ml centrifuge tube, add 2% CTAB600μl and 10μl mercaptoethanol; ②65 ℃ water bath for 15 min Add 500μl chloroform / isoamyl alcohol (24: 1) upside down to the liquid dark green; ④ centrifuge at 12 000 r / min for 3 min; ⑤ take the supernatant to -1.5 ml centrifuge tube, repeat steps ③, ④; ⑥ The supernatant was taken to -1.5 ml centrifuge tube, the tube pre-filled with 1 ml of 95% ethanol. ⑦ for 2 h at room temperature; ⑦ centrifuge at 12 000 r / min for 3 min; ⑧ discard the supernatant, wash the pellet with 70% ethanol (about 4400 μl) and dry in a vacuum desiccator. After drying plus low temperature preservation; ⑨ when used plus 100μl ultrapure water to dissolve. -25 ℃ save. Extract 50 μl of ultrapure water from the extracted DNA sample. After mixing, UV extinction was measured in a DU (R) 800 spectrophotometer using a quartz cuvette, which was calculated based on its absorbance at 260 nm and 280 nm DNA yield, according to A 260 (260) / A 280 (280) to determine the purity of DNA samples. The DNA sample concentration (μg / μl) is: UV extinction at 260 nm × Nucleic acid dilution × 50/1 000. Pure DNA sample A 260 / A 280 UV extinction value should be 1.8, A 260 / A 230 value should be greater than 2.0. Values of A 260 (260) / A 280 (280) greater than 1.9 indicate RNA contamination and less than 1.6 indicate protein or phenolic contamination in the sample. The value of A 260 / A 230 is less than 2.0, indicating that there are residual salts and small molecule impurities in the solution, such as nucleotides, amino acids, phenols and the like. The ferment DNA extracted by the conventional CTAB method and the modified CTAB method were respectively tested by agarose gel electrophoresis to verify the quality of extracted DNA. [Result] The ferment DNA extracted by the modified CTAB method had a brighter main band (genomic DNA) and distinct distinction with other hybrid bands, indicating that DNA degradation was less and DNA yield and purity were greatly improved. [Conclusion] The improved CTAB extraction method can extract high quality fern DNA.