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建立乌头叶片DNA甲基化MSAP分析方法,并对不同形状的乌头叶片DNA甲基化修饰位点进行MSAP分析。以花叶型、艾叶型乌头不同部位叶片为实验材料,考察乌头叶片MSAP双酶切反应时间,筛选适宜的选择性扩增引物,并通过计算6%丙烯酰胺凝胶电泳图泳道和条带统计分析叶片甲基化修饰差异。结果 150 ng乌头叶片DNA MSAP双酶切反应体系在37℃条件下,酶切16 h较充分;使用25对筛选出的适宜性引物进行乌头基因组的MSAP选择性扩增,总共获得273个电泳条带,其中无甲基化或单链内甲基化228条,双链内甲基化条带27条,单链外甲基化条带18条,总甲基化率达16.48%;花叶型与艾叶型乌头总甲基化率稍有差异,分别为15.36%,14.34%,并各自获得8条、6条基因组甲基化修饰差异的核苷酸片段,占其总条带数的3.00%,2.26%。通过研究可为乌头植株的分子鉴定、良种选育与栽培,以及乌头的遗传演化等提供新思路。
The DNA methylation MSAP analysis method was established and the MSAP analysis of DNA methylation modification sites in different shapes of Aconite leaves was established. The leaf segments of mosaic-type and leaf-shaped aconite were used as experimental materials to investigate the double digestion reaction time of MSAP. The suitable selective amplification primers were screened, and the 6% acrylamide gel electrophoresis pattern With statistical analysis of leaf methylation modification differences. Results The MSAP double digestion system of 150 ng aconite leaf DNA was digested at 37 ℃ for 16 h, and 25 pairs of suitable primers were selected for MSAP selective amplification of aconite genome. A total of 273 Electrophoresis bands, of which 228 were unmethylated or intramethylated, 27 were double - methylated and 18 were single - methylated. The total methylation rate reached 16.48%. There was a slight difference in the total methylation rate between mosaic type and Ai leaf type aconitum, which were 15.36% and 14.34%, respectively. Eight and six genomic DNA fragments with different methylation differences were accounted for 3.00% of the numbers, 2.26%. Through the research, we can provide new ideas for the molecular identification of Aconitum plants, breeding and cultivation of elite seeds, and the genetic evolution of Aconitum nigrum.