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目的探索一种适用于柴胡药材干根DNA提取的方法,为实现以分子标记方法辨别柴胡药材奠定基础。方法比较研究了分别用CTAB法、SDS法和高盐低pH法等3种方法提取柴胡样品基因组DNA。柴胡药材干根经过PVP以及TNE缓冲液进行不同的预处理,以CTAB法抽提DNA。以EcoRⅠ/MseⅠ双酶切琼脂糖凝胶电泳及RAPD扩增检测提取DNA的质量。采用NTSYS-pc软件计算Jaccard遗传相似系数,以非加权配对算术平均数法(UPGMA)建立聚类图。结果常规DNA提取方法提取药材干根DNA溶液经4℃放置后,黏稠并褐变,严重影响酶切和RAPD扩增。样品预处理优化条件是研磨时加入3%PVP,TNE缓冲液于0℃浸提2次,每次30min。以该优化的预处理方法处理6个柴胡药材干根样品,提取DNA条带清晰,酶切完全,RAPD扩增图谱清晰稳定,共获得有效扩增条带28条,其中多态性条带为20条,占71.43%。聚类分析表明,6个栽培品中,原产地为山西灵丘外地引种(LQWY)和甘肃陇西(LX)、山西灵丘(LQ)和山西方山(FSH)的亲缘关系较近,陕西商洛(SHL)和其他5个栽培品的亲缘关系较远。结论柴胡药材干根经过预处理后,可采用常规的DNA提取方法抽提DNA,所提DNA适合于酶切、RAPD等分子标记分析。
Objective To explore a suitable DNA extraction method for Radix Bupleuri to establish the foundation for molecular identification of Radix Bupleuri. Methods The genomic DNA of Bupleurum chinense Bupleurum samples were extracted by CTAB method, SDS method and high salt and low pH method respectively. Radix Bupleuri root dry through PVP and TNE buffer for different pretreatment, CTAB extraction of DNA. The quality of DNA was extracted by EcoRⅠ / MseⅠ double enzyme digestion agarose gel electrophoresis and RAPD amplification. The Jaccard genetic similarity coefficient was calculated by NTSYS-pc software and the non-weighted matching arithmetic mean (UPGMA) method was used to establish the clustering graph. Results Conventional DNA extraction method Extraction of dried roots DNA solution after 4 ℃ placed, sticky and brown, seriously affect the enzyme digestion and RAPD amplification. Sample pretreatment optimization conditions are added 3% PVP grinding, TNE buffer leaching 2 times at 0 ℃, each 30min. With the optimized pretreatment method, the roots of six Bupleurum samples were processed and the DNA bands were extracted and the DNA fragments were completely digested. The RAPD amplification pattern was clear and stable, and a total of 28 effective amplification bands were obtained. The polymorphic bands For 20, accounting for 71.43%. Cluster analysis showed that among the six cultivars, the genetic relationship between LQWY and LX, LQ and FSH in Shanxi origin was closer, (SHL) and other five cultivars are more distantly related. Conclusion After the roots of Bupleurum chinense have been pretreated, DNA can be extracted by conventional DNA extraction methods. The DNA is suitable for restriction analysis, RAPD and other molecular marker analysis.