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目的以核糖体转录间隔区(rDNA ITS)序列为分子标记,对钩藤属的2种常用中药材毛钩藤Uncariahirsute和无柄果钩藤U.sessilifructus进行遗传分析,阐明钩藤属内不同种间的分子系统发育关系。方法通过改良CTAB法对毛钩藤和无柄果钩藤进行总DNA提取,利用通用引物对rDNA ITS序列进行PCR扩增,RFLP分析和序列测定,用PAUP软件进行系统发育分析,构建最大简约树。结果获得毛钩藤和无柄果钩藤的限制性内切酶(MspⅠ&HaeⅢ)酶切图谱以及rDNA ITS区的完整序列,其序列长度均为718 bp。结论通过基于rDNAITS序列进行RFLP分析、序列测定和系统发育树构建的分子生物学方法,能够对钩藤属植物进行准确的分子鉴定,为钩藤属中药材的种类鉴定和种间的分类地位提供分子生物学依据。
OBJECTIVE: To analyze the genetic variations of Uncariahirsute and U. sessilifructus, two commonly used Chinese medicinal materials, Uncaria, using the rDNA ITS sequence as a molecular marker. Between the molecular phylogenetic relationships. Methods The total DNA was extracted from Cynanchum paniculata and Procambarus viridis by modified CTAB method. The rDNA ITS sequences were amplified by PCR, RFLP and sequenced using general primers. Phylogenetic analysis was performed with PAUP software to construct the largest parsimony tree. Results The sequence of restriction endonuclease (MspⅠ & HaeⅢ) and complete sequence of rDNA ITS region were obtained, and their sequence lengths were 718 bp. Conclusion Based on the rDNA ITS sequences, RFLP analysis, sequence analysis and phylogenetic tree construction of molecular biology methods can be used to accurately identify Uncaria plants and to identify the species and taxonomic status of the genus Uncaria. Molecular biology basis.